PrimePCR Assays, Panels, and Controls for Real-Time PCR Instruction Manual

PrimePCR Assays, Panels, and Controls for Real-Time PCR Instruction Manual
PrimePCR™ Assays, Panels, and
Controls for Real-Time PCR
Instruction Manual
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Contents
Chapter 1Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
PrimePCR Assays, Panels, and Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Shipping and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Reagents and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Tips for Success. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Chapter 2
Gene Expression Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Overview: Real-Time PCR Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Step 1: Isolate RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Step 2: Synthesize cDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Preamplification (Optional). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Step 3: Prepare Real-Time PCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Step 4: Cycle in Real-Time PCR Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Step 5: Analyze Gene Expression Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Chapter 3
Interpretation of Experimental Control Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Positive PCR Control Assay (PCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Reverse Transcription Control Assay (RT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
DNA Contamination Control Assay (gDNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
RNA Quality Assay (RQ1 and RQ2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter 4
DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Chapter 5
CFX Manager™ Software Quick Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
PrimePCR Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Data Analysis for Gene Expression Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Chapter 6
Reference Gene Selection and Reference Gene Panels. . . . . . . . . . . . . . . . . . . . . . . . . . 25
Chapter 7
MIQE Compliance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Chapter 8
Frequently Asked Questions (FAQs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Chapter 9Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Chapter 10
Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
PrimePCR Assays, Panels, and Controls for Real-Time PCR | iii
iv | PrimePCR Assays, Panels, and Controls for Real-Time PCR
1 Introduction
In 2012, Bio-Rad’s PrimePCR™ assays set a new standard for real-time PCR gene
expression analysis. We collaborated with leaders in real-time PCR to design, optimize, and
experimentally validate every primer pair. Products are available as individual SYBR® Green
assays, individual probe assays, pathway and disease panels, and custom-configured
plates. Reference gene and experimental control assays are also available. Assays are
validated for use with Bio-Rad’s amplification reagents. All PrimePCR assays and panels can
be ordered online at www.bio-rad.com/primepcr.
PrimePCR Assays, Panels, and Controls
SYBR® Green Assays
Transcriptome-wide assays are available for gene expression analysis. Individual assays are
provided as a 20x stock solution of 200, 1,000, or 2,500 reactions. Each PrimePCR assay
provides a Unique Assay ID, which can be easily tracked and reported in publications.
Probe Assays
5' exonuclease probe assays are available for gene expression analysis. Each assay can be
labeled with a FAM, HEX, Tex615, Cy5, or Cy5.5 fluorophore. Individual assays are provided
as a 20x stock solution in 500, 1,000, or 2,500 reactions. Each PrimePCR assay provides a
Unique Assay ID, which can be easily tracked and reported in publications.
Predesigned Pathway and Disease Panels
A wide range of pathway- or disease-specific gene panels are available in 96- or 384-well
plates. Primer pairs are lyophilized in the wells on the plate. Plates are designed for use with
SYBR® Green detection chemistry and include experimental controls and reference assays.
Plates are available for all major real-time PCR instruments (see the list of compatible realtime instruments under Reagents and Equipment).
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 1
| 1
Custom Plates
96- and 384-well PCR plates can be user-designed with PrimePCR™ SYBR® Green assays.
Custom primer assays can also be added. Primer pairs are lyophilized in the wells on the
plate. Plates are available for all major real-time PCR instruments, and can be designed with
or without experimental controls.
PreAmp Assays
PrimePCR PreAmp assays are available for unbiased preamplification of target genes from
small quantities of cDNA. Each assay is provided as a 100x stock solution and corresponds
to a SYBR® Green or probe gene expression assay.
DNA Templates
Assay-specific synthetic DNA templates are designed to give a positive PCR result when
used with the corresponding SYBR® Green or probe assay. Templates are provided as a 20x
stock solution of 200 reactions.
Experimental Control Assays
Experimental control assays are available for RNA quality assessment, genomic DNA
contamination, reverse transcription, and PCR performance. Controls can be ordered as
individual assays or preplated on predesigned or custom PCR plates.
Reference Gene Assays
Commonly used reference gene assays are available to normalize for variation in the amount
of input messenger RNA (mRNA) among samples. Assays can be ordered as individual
assays or added to a custom plate. Predesigned reference gene panels are available in
96- and 384-well plates.
Droplet Digital™ PCR (ddPCR™) Probe Assays
Copy number variation and mutation detection assays have been expertly designed and
validated for use with the QX100™ and QX200™ Droplet Digital PCR platform. Probe assays
are labeled with a FAM or a HEX fluorophore, and are available in 200, 1,000, and 2,500
reactions. Please see the PrimePCR ddPCR product inserts for detailed procedure and
assay information.
Shipping and Storage
PrimePCR assays and plates are shipped at room temperature. Upon receipt, store product
at 4°C (up to 12 months). For long-term storage, store at –20°C.
If storing assay at –20°C, prevent oligonucleotide degradation by aliquoting the total volume
into smaller volumes to minimize the number of freeze-thaw cycles.
cDNA synthesis kits and supermixes are shipped on dry ice and should be stored at –20°C.
Refer to individual product manuals for more details.
2 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
Reagents and Equipment
Consumables and Equipment Needed for Gene Expression Analysis
■■
cDNA synthesis kit
■■
Centrifuge
■■
Gene expression analysis software
■■
Microfuge tubes
■■
Nuclease-free pipet tips and tubes
■■
Nuclease-free water
■■
Optical seals
■■
PCR plates or strips (if not using a preplated panel)
■■
Pipets — single channel and multichannel
■■
Purified RNA samples
■■
Real-time PCR detection system
■■
SYBR® Green or probe supermix
■■
■■
TE buffer (pH 7.5, nuclease-free) — use for dilution of reverse transcription (RT)
control template
TE buffer with 10 ng/ml tRNA (pH 8.0, nuclease-free) — use for standard curve dilutions
Guaranteed Performance
PrimePCR assays are guaranteed to perform with the following qPCR reagents:
*
■■
iScript™ advanced cDNA synthesis kit for RT-qPCR*
■■
iScript cDNA synthesis kit
■■
iScript reverse transcription supermix for RT-qPCR
■■
SsoAdvanced™ PreAmp supermix
■■
SsoAdvanced™ SYBR® Green supermix*
■■
SsoAdvanced universal probes supermix
■■
SsoAdvanced™ universal SYBR® Green supermix
■■
iTaq™ universal probes supermix
■■
iTaq™ universal SYBR® Green supermix
Reagents used for validation report
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 3
Compatible Real-Time PCR Instruments
PrimePCR assays and panels are available for the following real-time PCR detection systems:
Bio-Rad
CFX96™, CFX384™, CFX96 Touch™, CFX384 Touch™, CFX Connect™, Chromo4™, iQ™, iQ™5,
MyiQ™, MyiQ™2, Opticon™, Opticon™ 2
ABI
7300, 7500 (standard and Fast), 7900 (standard and Fast, 96- and 384-well), ViiA 7
(standard and Fast, 96- and 384-well), QuantStudio (standard and Fast, 96- and 384-well),
StepOnePlus
Stratagene Mx
Eppendorf realplex
Roche LightCycler 480 (96- and 384-well)
Important: Verify instrument and fluorophore compatibility.
Tips for Success
For a complete description of how to optimize a gene expression experiment, please visit
the gene expression workflow section of our Applications and Technologies pages at
www.bio-rad.com/en-us/applications-technologies/gene-expression-analysis.
Ensure High RNA Quality and Quantity
To obtain optimal real-time PCR results, start with high-quality RNA from all samples.
RNases, salts, proteins, and other contaminants can compromise the integrity of RNA
samples and may impact the performance of downstream enzymes used in reverse
transcription and PCR reactions.
■■
Decontaminate the workspace of RNase, salt, and protein sources
■■
Limit the number of freeze-thaw cycles of RNA sample, ideally to one cycle
■■
Use screw-cap tubes for long-term RNA storage at –80°C
■■
Use nuclease-free plastics and solutions
■■
Determine concentration and purity of RNA prior to gene expression experiment
■■
Mass normalize RNA samples by dilution with nuclease-free water prior to the reverse
transcription reaction
4 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
Prevent and Monitor Genomic DNA Contamination
Genomic DNA (gDNA) present in real-time PCR experiments can cause inaccurate gene
expression quantification. Although PrimePCR assays were designed for intron-spanning
regions when possible, elimination of gDNA contamination is a best practice for high-quality
qPCR results.
■■
■■
Perform a DNA digestion step with DNase during RNA purification
Include a gDNA experimental control to determine if gDNA is present (see DNA
Contamination Control Assay in Step 3 of Chapter 2, Gene Expression Protocols)
Select Appropriate Reference Genes
Choosing an appropriate reference gene(s) to normalize for variation in the amount of
input messenger RNA (mRNA) among samples is important for accurate gene expression
analysis. Consult Chapter 6, Reference Gene Selection and Reference Gene Panels, to learn
how to determine a stably expressed gene target for your samples of interest.
Calibrate the Real-Time PCR Instrument for Dyes and Fluorophores
Prior to starting a qPCR experiment, verify that the real-time PCR instrument is compatible
and calibrated correctly for the dyes and fluorophores chosen. CFX Manager™ software
has a Dye Calibration Wizard with an easy guide to calibration (Tools > Dye Calibration
Wizard). Bio-Rad’s CFX instruments are currently calibrated with pure dyes to correspond
to PrimePCR fluorophore options before installation. Refer to the instrument manual to
determine calibration settings, and calibrate accordingly if necessary.
Fluorophore/Dye
Excitation, nm
Emission, nm
®
SYBR Green
494
521
5' FAM
495
520
5' HEX
538
555
5' TEX615
596
613
5' Cy5
648
668
5' Cy5.5
685
706
Use Optical PCR Plate Seals
Use optical seals for accuracy when collecting real-time PCR data. Adhesive seals and heat
seals are available options. Bio-Rad recommends using the PX1™ PCR plate sealer with
optical heat seals for consistently sealed PCR plates. Seals must fully enclose the PCR plate
to prevent evaporation from the wells during thermal cycling. Evaporation is identified by
analyzing technical repeats of data — specifically, if a well near the edge of the plate yields
variable data, evaporation has likely occurred.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 5
Determine the Threshold Setting
Determining placement of the threshold line is important for accurate quantification of gene
expression levels. For precise comparisons of gene expression levels between samples,
determine Cq values for a gene only when the threshold line has been defined uniformly
for all samples being analyzed. While the threshold can be set manually or automatically
using the instrument’s software, it is important that the same numerical threshold value be
used across all samples, whether they are on the same or multiple plates. The threshold is
typically placed at least 10 standard deviations above the average baseline fluorescence
level where the PCR reaction is undergoing exponential amplification. As an example,
300 rfu was used as the uniform threshold line setting for all PrimePCR assay validation
experiments on a CFX384 instrument.
Multiplex Assay Considerations
PrimePCR probe assays have been optimized to work under the same thermal cycler
conditions, allowing for multiple assays to be used in the same well with different
fluorophores. To ensure data generated in a multiplex reaction are equivalent to data
generated in a singleplex reaction, it is imperative to evaluate the assay performance in
both multiplex and singleplex reactions. It is also important to understand the expression
level of your target sequences, as this will impact the multiplex optimization method.
Consult the SsoAdvanced Universal Probes Supermix Instruction Manual for more details
on multiplexing.
Below is a brief list of considerations when conducting multiplex reactions:
■■
Determine expression levels of gene targets
■■
Use brighter fluorophores for lower expressing targets
■■
Compare standard curve of singleplex reaction to multiplex reaction
■■
Efficiency of multiplex reaction must be similar to minimize amplification bias
■■
Target abundance should not vary greatly between multiplex assays
6 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
2 Gene Expression Protocols
This chapter provides protocols for RNA isolation, cDNA synthesis, real-time PCR reaction
setup (in each PrimePCR™ format), and the real-time PCR instrument run. Please follow the
appropriate protocol for the format of your experiment (individual assays, 96- or 384-well
plates). Remember to set up controls alongside reverse transcription and PCR reactions.
Overview: Real-Time PCR Workflow
Step 1: Isolate RNA
■■
Aurum™ total RNA mini kit
■■
Aurum total RNA fatty and fibrous kit
Step 2: Synthesize cDNA
■■
iScript™ family of reverse transcription reagents
Optional: Prepare Preamplification Reaction
−− PrimePCR PreAmp assays
−− SsoAdvanced PreAmp supermix
Step 3: Prepare Real-Time PCR Reaction
■■
PrimePCR assays and panels
■■
SsoAdvanced™ universal supermixes
Step 4: Cycle in Real-Time PCR Instrument
■■
CFX real-time PCR detection systems
Step 5: Analyze Gene Expression Data
■■
CFX Manager™ software
■■
PrimePCR analysis software
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 7
Step 1: Isolate RNA
1. Isolate and purify the RNA sample(s) using Aurum isolation kits or a similar product.
The Aurum total RNA mini kit (catalog #732-6820) and the Aurum fatty and fibrous tissue
kit (catalog #732-6830) enable RNA isolation from a wide range of cells and tissues and
include a DNase digestion step to ensure DNA-free total RNA. Refer to the instruction
manual for the detailed protocol.
2. Analyze the RNA sample for integrity and purity.
a. Use Bio-Rad’s Experion™ automated electrophoresis system, the Agilent Bioanalyzer,
or similar system to evaluate the integrity of the RNA sample. For gene expression
analysis, ensure that the RNA quality indicator (RQI) or RNA integrity number (RIN)
values are similar between samples to yield accurate qPCR results. An RQI value of
10 indicates intact RNA, whereas a value of 1 indicates degraded RNA.
b. Use an agarose gel to assess RNA integrity if the above systems are not available.
High-quality eukaryotic RNA yields two clean peaks, 18S and 28S. Degraded RNA
appears as a smear on the gel.
c. To assess purity, evaluate the following spectrophotometer readings:
A260/A280 > 2.0 for pure RNA (assesses DNA contamination)
A260/A230 ~2.0 for pure RNA (assesses protein contamination)
Lower ratios indicate the presence of contaminants such as salts, carbohydrates,
peptides, proteins, phenols, and guanidine thiocyanate.
3. Assess total RNA concentration using a SmartSpec™ Plus spectrophotometer or similar
instrument.
4. Normalize RNA concentration across samples by diluting in nuclease-free water.
5. Store RNA sample(s) at –80°C in single-use aliquots and at equivalent concentrations.
Step 2: Synthesize cDNA
Use the iScript family of reverse transcription reagents for cDNA synthesis. The iScript
advanced cDNA synthesis kit was used during wet-lab validation of PrimePCR assays. In a
single 20 µl reaction, this two-tube kit provides increased data throughput and the widest
possible linear dynamic range through its loading capacity of up to 7.5 µg total RNA.
1. Thaw kit contents and normalized RNA samples on ice. Mix components thoroughly,
centrifuge briefly to collect solutions at the bottoms of tubes, and then store on ice.
2. Use the same amount of RNA in each cDNA synthesis reaction. Typically 1–5 µg total
RNA is sufficient, but may be adjusted depending on abundance of target transcript.
Reagents
Description
5x iScript advanced reaction mix (red cap)
5x reaction mix with dNTPs, oligo(dT), random primers, buffer,
MgCl2, enhancers, and stabilizers
iScript advanced reverse transcriptase
(orange cap)
iScript MMLV-RT (RNase H+) and RNase inhibitor
Nuclease-free water
8 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
3. For each RNA sample, prepare a cDNA synthesis reaction on ice.
4. If using the PrimePCR RT control assay, dilute the lyophilized RT control assay template
in 200 µl nuclease-free TE (pH 7.5) or the same reagent used to dilute RNA samples.
Keep the template on ice. It will degrade similar to RNA samples if exposed to multiple
freeze-thaw cycles, ambient temperatures, or RNases. When preparing the cDNA
synthesis reaction, add 1 µl of the diluted RT control template.
Component
Volume per Reaction
5x iScript advanced reaction mix
4 μl
iScript advanced reverse transcriptase
1 μl
RNA (100 fg to 7.5 µg)*
Variable
RT control assay template
1 μl
Nuclease-free water
Variable
Total volume
20 μl
* It may be necessary to optimize the amount of RNA input due to differences in target gene
expression levels and sample availability. See step 6 for an example calculation.
5. Incubate the complete reaction mix in a thermal cycler using the protocol below:
Reverse transcription: 30 min at 42°C
RT inactivation: 5 min at 85°C
6. Dilute the cDNA reaction to the desired volume for qPCR reactions. The ideal range of
input cDNA per qPCR reaction is 1–25 ng. Altering the amount of input RNA or the level of
cDNA dilution can optimize gene expression results for a given collection of genes. Below
is a step-by-step example calculation from starting RNA input through amount of cDNA
per qPCR reaction used for a PrimePCR 96-well panel with 96 unique gene targets:
1 µg RNA input per 20 µl RT reaction = 50 ng/µl
Dilute cDNA to 100 µl = 10 ng/µl
Use 1 µl diluted cDNA per 20 µl qPCR reaction = 10 ng cDNA per reaction
Prepare and Run Preamplification Reaction (Optional)
For the unbiased amplification of small quantities of cDNA, follow the preamplification
protocol prior to preparing the real-time PCR reaction.
Prepare Preamplification Assay Pool with PrimePCR PreAmp Assays
1. Add 5 µl of each PreAmp assay (up to 100 assays) to a microcentrifuge tube.
2. Bring total volume of assay pool up to 500 µl with nuclease-free water.
3. Mix thoroughly, briefly centrifuge, and store on ice.
4. Use 5 µl of the assay pool in a 50 µl reaction.
Note: Assay pools are stable at 4°C for up to 30 days and at –20°C for up to 1 year.
PrimePCR control assays are currently not compatible with preamplification.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 9
Prepare Preamplification Reaction
1. Thaw SsoAdvanced PreAmp supermix and cDNA samples. Mix thoroughly by inversion
or by a 15 sec vortex. Centrifuge briefly to collect solutions at the bottom of each tube
and then store on ice and protected from light.
2. Prepare preamplification reaction mix on ice. Good pipetting practice must be employed
to ensure assay precision and accuracy.
Component
Volume per Reaction
Final Concentration
2x SsoAdvanced PreAmp supermix
25 μl
1x
PrimePCR PreAmp pool
5 μl
1x
cDNA sample
Variable
250 ng–100 pg
Nuclease-free water
Variable
—
50 μl
—
Total volume
3. Mix the reaction mix thoroughly to ensure homogeneity and dispense into a PCR tube or
into the wells of a PCR plate.
4. Load the PCR tubes or plate into a thermal cycler and start the PCR run with the
following thermal cycling protocol.
Step
Temperature
Time
# of Cycles
Activation
95°C
3 min
1
Denaturation
95°C
15 sec
10–12
Annealing/extension
58°C
4 min
10–12
4°C
∞
1
Hold
5. After run completion, the preamplification reaction can be stored at –20°C for up to
12 months or at 4°C for up to 72 hr.
6. The completed preamplification reaction should be diluted to a minimum of 1:5 with
TE buffer. However, a larger dilution volume may be required, depending on the number
of assays planned for downstream qPCR and the number of technical replicates.
For example, a 1:15 dilution will provide enough volume for 100 qPCR assays using
technical triplicates and 2 µl per reaction.
100 qPCR assays in triplicate = 100 assays x 3 replicates x 2 µl = 600 µl needed
1:15 preamplification reaction dilution = 750 µl
150 µl excess
7. Use 2 µl of the dilution per 20 µl qPCR reaction or 1 µl per 10 µl qPCR reaction for a
96- or a 384-well plate, respectively.
Note: When using SsoAdvanced universal SYBR® Green supermix for qPCR after
preamplification, run the activation and denaturation steps at 98°C if delayed Cq values or
decreased efficiency occur.
10 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
Step 3: Prepare Real-Time PCR Reaction
Protocols for preparing real-time PCR reaction mix are described according to format
(96-well plates, 384-well plates, or individual assays). Protocols for PrimePCR control
assays are incorporated into the reaction setup. Please refer to Chapter 3 for interpretation
of experimental control assays.
Pathway/Disease Panel or Custom Plate — 96-Well Format
Primers are lyophilized in each well; therefore, add all remaining components to the PCR
reaction mix.
1. Thaw SsoAdvanced™ universal SYBR® Green supermix, cDNA samples, and positive
PCR control DNA template (if a PCR control assay is on the plate). Mix thoroughly by
inversion or a 15 sec vortex, centrifuge briefly to collect solutions at the bottoms of
tubes, and then store on ice and protected from light.
2. Allow the PrimePCR plate to come to room temperature prior to removing the seal.
3. Dilute each cDNA sample to a total volume of 100 µl if preparing a 96-well plate with
unique targets.
4. Prepare (on ice or at room temperature) enough reaction mix for all qPCR reactions by
adding all required components. Scale up proportionally for multiple reactions.
CFX Manager™ software has an easy-to-use master mix calculator that provides
scale-up calculations for your experiment.
Component
Volume per Reaction
Final Concentration
Dried in well
1x
10 μl
1x
cDNA sample
1–4 μl*
100 ng–100 fg*
Nuclease-free water
Variable
—
20 μl
—
20x PrimePCR assay
2x SsoAdvanced™ universal SYBR® Green
supermix
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume (4 µl) of cDNA
sample to avoid excessive carryover of PCR inhibitors.
5. Transfer 20 µl of the PCR reaction mix into each well.
6.Optional: Add 1 µl PCR control assay template into the appropriate well (total volume
will be 21 µl — additional volume will not affect the qPCR reaction).
7. Seal the plate with an optical seal. Optional: briefly centrifuge to remove bubbles (4,000 rpm
for 2 min at room temperature).
8. Load the PCR plate into the real-time PCR instrument.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 11
Pathway/Disease Panel or Custom Plate — 384-Well Format
Primers are lyophilized in each well; therefore, add all remaining components to the PCR
reaction mix.
1. Thaw SsoAdvanced™ universal SYBR® Green supermix, cDNA samples, and positive
PCR control DNA template (if a PCR control assay is on the plate). Mix thoroughly by
inversion or a 15 sec vortex, centrifuge briefly to collect solutions at the bottoms of
tubes, and then store on ice and protected from light.
2. Allow the PrimePCR plate to come to room temperature prior to removing the seal.
3. Dilute each cDNA sample to a total volume of 400 µl if preparing a 384-well plate with
unique targets.
4. Prepare (on ice or at room temperature) enough reaction mix for all qPCR reactions by
adding all required components. Scale up proportionally for multiple reactions.
CFX Manager software has an easy-to-use master mix calculator that provides easy
scale-up calculations for your experiment.
Component
Volume per Reaction
Final Concentration
Dried in well
1x
5 μl
1x
cDNA sample
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
—
10 μl
—
20x PrimePCR assay
2x SsoAdvanced™ universal SYBR® Green
supermix
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample
to avoid excessive carryover of PCR inhibitors.
5. Transfer 10 µl of the PCR reaction mix into each well.
6.Optional: Add 0.5 µl PCR control assay template into the appropriate well (total volume
will be 10.5 µl — additional volume will not affect the qPCR reaction).
7. Seal the plate with an optical seal. Optional: briefly centrifuge to remove bubbles (4,000 rpm
for 4 min at room temperature).
Note: Be careful to not introduce bubbles during pipetting steps, since it is difficult to
remove bubbles from 384-well plates.
8. Load the PCR plate into the real-time PCR instrument.
Individual Assays
Protocols for primer assays, probe assays, and control assays follow. PrimePCR individual
assays come ready to use as a 20x stock solution.
12 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
SYBR® Green Assay
1. Thaw SsoAdvanced™ universal SYBR® Green supermix, PrimePCR™ SYBR® Green
assay, cDNA samples, and positive PCR control (if used) to room temperature. Mix
thoroughly by inversion or a 15 sec vortex, centrifuge briefly to collect solutions at the
bottoms of tubes, and then store on ice and protected from light.
2. Prepare (on ice or at room temperature) enough reaction mix for all qPCR reactions
by adding all required components. PrimePCR™ SYBR® Green assays are provided
as a 20x stock solution. Scale up proportionally for technical replicates. CFX Manager
software has an easy-to-use master mix calculator that provides easy scale-up
calculations for your experiment.
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
20x PrimePCR assay
1 μl
0.5 μl
1x
2x SsoAdvanced universal SYBR® Green
supermix
10 μl
5 μl
1x
cDNA sample
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample
to avoid excessive carryover of PCR inhibitors.
3. Transfer the appropriate volume of the PCR reaction mix into each tube or well: 20 µl for
a 96-well plate, or 10 µl for a 384-well plate.
4. Optional: Set up control reactions to determine sample quality and PCR and RT
reaction performance.
5. Seal the plate with an optical seal. Optional: briefly centrifuge to remove bubbles (4,000 rpm
for 2 min at room temperature).
6. Load the PCR plate (or tubes) into the real-time PCR instrument.
Probe Assay
1. Thaw SsoAdvanced universal probes supermix, PrimePCR probe assay, cDNA samples,
and positive PCR control (if used) to room temperature. Mix thoroughly by inversion or
a 15 sec vortex, centrifuge briefly to collect solutions at the bottoms of tubes, and then
store on ice and protected from light.
2. Prepare (on ice or at room temperature) enough reaction mix for all qPCR reactions
by adding all required components. PrimePCR probe assays are provided as a 20x
stock solution. Scale up PCR reaction mix proportionally for technical replicates.
CFX Manager software has an easy-to-use master mix calculator that provides easy
scale-up calculations for your experiment.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 13
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
20x PrimePCR assay
1 μl
0.5 μl
1x
2x SsoAdvanced universal probes supermix
10 μl
5 μl
1x
cDNA sample
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample
to avoid excessive carryover of PCR inhibitors.
3. Transfer the appropriate volume of the PCR reaction mix into each tube or well: 20 µl for
a 96-well plate, or 10 µl for a 384-well plate.
4. Optional: Set up control reactions to determine sample quality and PCR and RT
reaction performance.
5. Seal the plate with an optical seal. Optional: briefly centrifuge to remove bubbles (4,000 rpm
for 2 min at room temperature).
6. Load the PCR plate (or tubes) into the real-time PCR instrument.
Positive PCR Control Assay (PCR)
1. For each cDNA sample, prepare a positive PCR control reaction mix (template and assay
in the same tube).
Component
20x PrimePCR positive PCR control assay
2x SsoAdvanced supermix
Volume per Reaction
96-Well
384-Well
1 μl
0.5 μl
Final Concentration
1x
10 μl
5 μl
1x
cDNA sample
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample
to avoid excessive carryover of PCR inhibitors.
2. Follow the remainder of the PrimePCR cycling protocol.
3. Assess performance of the qPCR reaction (see Chapter 3, Interpretation of Experimental
Control Assays).
Reverse Transcription Control Assay (RT)
1. Resuspend lyophilized RT control assay template in 200 µl nuclease-free TE (pH 7.5) or the
same reagent used to dilute RNA samples. Keep template on ice. It will degrade similar to
RNA samples if exposed to multiple freeze-thaw cycles, ambient temperatures, or RNases.
14 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
2. For each RNA sample, include 1 µl of the control RNA template in each 20 µl cDNA
synthesis reaction, and proceed with the reverse transcription reaction.
3. Once cDNA is synthesized and diluted to working stock, prepare a reverse transcription
control reaction mix.
Component
Volume per Reaction
20x PrimePCR RT control assay
2x SsoAdvanced supermix
96-Well
384-Well
1 μl
0.5 μl
Final Concentration
1x
10 μl
5 μl
1x
cDNA sample with RT control template
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA
sample to avoid excessive carryover of PCR inhibitors.
4. Follow the remainder of the PrimePCR cycling protocol.
5. Assess performance of the RT reaction (see Chapter 3, Interpretation of Experimental
Control Assays).
DNA Contamination Control Assay (gDNA)
1. For each cDNA sample, prepare a DNA contamination control reaction mix.
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
20x PrimePCR gDNA control assay
1 μl
0.5 μl
1x
2x SsoAdvanced supermix
10 μl
5 μl
1x
cDNA sample with RT control template
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample
to avoid excessive carryover of PCR inhibitors.
2. Follow the remainder of the PrimePCR cycling protocol.
3. Assess performance of the RT reaction (see Chapter 3, Interpretation of Experimental
Control Assays).
RNA Quality Assay (RQ1 and RQ2)
1. For each cDNA sample, prepare RQ1 and RQ2 RNA quality assay reaction mixes.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 15
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
20x PrimePCR RQ1 assay
1 μl
0.5 μl
1x
2x SsoAdvanced supermix
10 μl
5 μl
1x
cDNA sample
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
Total volume
20 μl
10 μl
—
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
20x PrimePCR RQ2 assay
1 μl
0.5 μl
1x
2x SsoAdvanced supermix
10 μl
5 μl
1x
cDNA sample
1–4 μl*
0.5–2 μl*
100 ng–100 fg*
Nuclease-free water
Variable
Variable
—
20 μl
10 μl
—
Total volume
* Calculated during Step 2, cDNA synthesis. Avoid adding >20% reaction volume of cDNA sample to
avoid excessive carryover of PCR inhibitors.
2. Follow the remainder of the PrimePCR cycling protocol.
3. Assess performance of the qPCR reaction (see Chapter 3, Interpretation of Experimental
Control Assays).
Step 4: Cycle in Real-Time PCR Instrument
Load the PCR tubes or plate on the real-time PCR instrument and program the thermal
cycling protocol according to the following table. Start the real-time PCR run.
Step
Temperature
Time
No. of Cycles
Activation
95°C
2 min*
1
Denaturation
95°C
5 sec
40
Annealing/Extension
60°C
30 sec
40
65–95°C
(O.5°C increments)
5 sec/step
1
Melt Curve**
* Activation can be reduced to 30 sec. Do not use a 10-min activation time with Bio-Rad supermixes.
** Melt curve step is for SYBR® Green analysis only.
Step 5: Analyze Gene Expression Data
Process your data and evaluate results using CFX Manager or other analysis software. See
Chapter 5, CFX Manager Quick Guide, or consult your instrument and software manuals for
detailed instructions. If PrimePCR control assays were used, refer to Chapter 3 to interpret
sample quality and reaction performance.
16 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
3 Interpretation of
Experimental Control Assays
PrimePCR™ experimental control assays are designed to help assess the quality of your
sample(s) and how this may affect the performance of the reverse transcription and qPCR
reactions. They work alongside any assay from any source. This chapter explains how
to interpret control assays. For control assay reaction setup, refer to the format (plate or
individual assay) in Chapter 2, Gene Expression Protocols.
Positive PCR Control Assay (PCR)
Purpose: The positive PCR control assay (PCR) targets a synthetic DNA template to
determine if samples contain inhibitors or other factors that may negatively affect your gene
expression results. The sequence of the synthetic DNA template is not present in the human
or mouse genome.
Designed to qualitatively assess:
■■
Performance of a qPCR reaction associated with a single sample
■■
Relative performance of the qPCR reactions associated with different samples
Format:
■■
■■
Individual assay — 200 reactions (20x stock solution includes primers and DNA template)
96-well or 384-well plate — primers are lyophilized in designated well(s) on the plate, and
the DNA template is provided in a separate tube
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 17
Interpretation:
■■
■■
Single sample — Cq ≥ 30 indicates poor PCR performance; will likely compromise gene
expression results
Two or more samples — designate one of your samples as the control. To determine the
ΔCq between the control sample and each remaining sample, use the following equation:
|(PCR Cq for control sample) – (PCR Cq for sample)| = ΔCq
ΔCq > 1 indicates samples differ in their impact on qPCR performance and may
compromise gene expression results.
Reverse Transcription Control Assay (RT)
Purpose: The reverse transcription (RT) control assay introduces a synthetic RNA template
into the cDNA synthesis reaction to evaluate the RT performance. The sequence of the
synthetic RNA template is not present in the human or mouse transcriptome.
Designed to qualitatively assess:
■■
■■
Performance of a reverse transcription reaction associated with a single sample
Relative performance of the reverse transcription reactions associated with different
samples
Format:
■■
■■
Individual assay — 200 reactions of 20x stock solution PrimePCR reverse transcription
control primer assay and 200 reactions lyophilized RNA template
96-well or 384-well plate — primers are lyophilized in designated well(s) on the plate,
and the lyophilized RNA template is provided in a separate tube
Interpretation:
■■
■■
Single sample — Cq ≥ 30 indicates poor reverse transcription reaction performance; will
likely compromise gene expression results
Two or more samples — designate one of your samples as the control. To determine the
ΔCq between the control sample and each remaining sample, use the following equation:
|(RT Cq for control sample) – (RT Cq for sample)| = ΔCq
ΔCq > 1 indicates that samples differ in their impact on reverse transcriptase
performance and may compromise gene expression results.
18 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
DNA Contamination Control Assay (gDNA)
Purpose: The DNA contamination control assay (gDNA) is a species-specific control assay
that targets a nontranscribed region of the genome.
Designed to qualitatively assess:
■■
■■
Whether genomic DNA (gDNA) is present in a sample at a level that may affect qPCR
results
Relative levels of gDNA contamination present in different samples to determine if qPCR
results may be affected
Format:
■■
Individual assay — 200 reactions (20x stock solution)
■■
96-well or 384-well plate — primers are lyophilized in designated well(s) on the plate
Interpretation:
Single sample:
■■
■■
Cq ≥ 35: indicates below single copy detection; no gDNA present
Cq < 35: indicates the sample is contaminated with gDNA and gene expression results
may be affected. The relative contribution of gDNA contamination to a sample’s signal
can be determined by comparing the Cq value for a given gene of interest (GOI) to the Cq
value for the DNA contamination control assay using the following equation:
|(GOI Cq) – (gDNA Cq)| = ΔCq
∆Cq
Percent Contribution
1
50%
2
25%
3
12.5%
4
6.25%
5
3.13%
6
1.56%
7
0.78%
Two or more samples: Designate a sample as the control. To determine the ΔCq between
the control sample and each remaining sample, use the following equation:
|(gDNA Cq for control sample) – (gDNA Cq for sample)| = ΔCq
■■
■■
ΔCq < 1: samples have similar levels of gDNA contamination; gDNA contamination will
likely have little to no effect on results
ΔCq ≥ 1: samples have different levels of gDNA contamination; gDNA contamination may
affect gene expression results
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 19
RNA Quality Assay (RQ1 and RQ2)
Purpose: The RNA quality assay is a pair of assays (RQ1 and RQ2) that target the same
transcript at different locations with different amplicon sizes. The RQ1 and RQ2 assays must
be used as a pair for each cDNA sample. Differences in Cq values can indicate whether
RNA degradation may be negatively impacting gene expression results.
Designed to qualitatively assess:
■■
Whether RNA integrity may adversely affect PCR results of a single sample
■■
Relative RNA integrity between samples to determine if qPCR results may be affected
Format:
■■
■■
Individual assay — 200 reactions (20x stock solution) of RQ1 RNA quality assay and
200 reactions (20x stock solution) of RQ2 RNA quality assay
96-well or 384-well plate — RQ1 and RQ2 primers are lyophilized in designated well(s)
on the plate
Interpretation:
■■
Single sample — to determine the ΔCq between the RQ1 and RQ2 assays, use the
following equation:
|(RQ2 Cq) – (RQ1 Cq)| = ΔCq
ΔCq ≤ 3.0: RNA degradation is minimal and will likely have little to no effect on gene
expression results
ΔCq > 3.0: RNA integrity may compromise gene expression results
■■
Two or more samples — designate one of your samples as your control sample. To
determine the ΔΔCq between the control sample and each remaining sample, use the
following equation:
[ |(RQ2 Cq for designated control sample) – (RQ1 for designated control sample Cq)| ] –
[ |(RQ2 Cq for sample) – (RQ1 for sample Cq)| ] = ΔΔCq
ΔΔCq = 0 to 1.0: samples are similar in quality; will likely have little to no effect on
gene expression results
ΔΔCq = 1.0 to 2.0: RNA integrity differs; will likely have slight to moderate effects on
gene expression results
ΔΔCq > 2.0: RNA integrity differs; will likely have significant effects on gene
expression results
20 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
4 DNA Templates
PrimePCR™ DNA templates are single-stranded synthetic DNA templates that are
complementary to corresponding gene-specific PrimePCR assays. Each template is linked
to the corresponding assay with a Unique Assay ID. Templates for use with SYBR® Green
assays are 60 base pairs in length, and those for use with probe assays are 90 base pairs in
length. They are designed to give a positive real-time PCR result when used with the correct
PrimePCR assay and can also be used to generate a standard curve.
Format: individual template — 200 reactions (20x stock solution) of assay-specific template
(20 x 106 copies/µl)
Procedure:
1. For use as a qPCR control, prepare the reaction mix.
Component
Volume per Reaction
Final Concentration
96-Well
384-Well
1 μl
0.5 μl
10 μl
5 μl
1x
20x PrimePCR cDNA template for gene of interest
1 μl
0.5 μl
20 x 10 copies/μl
Nuclease-free water
8 μl
4 μl
—
20 μl
10 μl
—
20x PrimePCR assay for gene of interest
2x SsoAdvanced
™
supermix
Total volume
1x
6
2. Follow the PrimePCR cycling protocol and evaluate results to determine assay
performance.
Interpretation: When used as a qPCR control
■■
Cq < 30: assay performance is not affected
■■
Cq > 30: poor PCR performance; will likely compromise results
Standard Curve:
Perform a standard curve using a seven-point, tenfold serial dilution series from 20 million
copies to 20 copies. Prepare serial dilutions of a stock solution of 10 ng/ml tRNA in TE (pH 8.0).
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 21
22 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
5 CFX Manager
™
Quick Guide
Software
For detailed instructions, please refer to your CFX instrument manual or CFX Manager
software manual.
PrimePCR™ Run Setup
1. To begin a PrimePCR protocol, select the PrimePCR button from the Startup Wizard and
go to File > New > PrimePCR run, or drag and drop a PrimePCR run file* onto the main
window of CFX Manager.
2. Once a PrimePCR run has been selected, the Run Setup window opens in the Start Run
tab. The PrimePCR default protocol and plate layout are loaded.
3. For SYBR® Green or FAM experiments, select the SYBR/FAM scan mode. For probe
assays using other fluorophores (HEX and Tex615) or for multiplex experiments, open
the Plate tab and use the Edit Selected option to choose the specific fluorophores and
select the All Channels scan mode.
4. For SYBR® Green assays, select Include Melt Step in the Protocol tab; deselect it for
probe assays.
5. Once the plate and protocol are loaded, click Close Lid and Start Run.
*
PrimePCR run files for predesigned and custom plates can be downloaded from www.bio-rad.com/primepcr. Custom plates are
saved under My PrimePCR once configured and purchased. Predesigned panels have a Download Run File button on the Review
Plate page.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 23
Data Analysis for Gene Expression Experiments
1. Drag and drop the PrimePCR run file for a predesigned or custom plate before or after a
protocol run in order to populate the assay names for data analysis. Alternatively, open a
data file by selecting File > Open > Data file. If analyzing data from multiple plates, skip
to step 5 and set up a gene study.
2. The plate should contain sample and target information, as well as controls. Select
reference targets in Experiment Settings.
3. Once the above requirements are met for normalized gene expression analysis, the
Quantification and Quantification Analysis tabs of the Data Analysis window display a
table of Cq values, averages, standard deviations, and normalized values.
4. Bar charts, clustergrams, scatter plots, volcano plots, and heat maps are available under
the Gene Expression tab.
5. Create a gene study to compare gene expression data from one or more real-time PCR
experiments using an inter-run calibrator to normalize between experiments.
a. Go to File > New > Gene Study.
b. A Gene Study window opens. To add data files for the gene study, select File > Add
Data File and choose the .pcrd data files you would like to analyze.
c. CFX Manager software automatically attempts an inter-run calibration (IRC) to
normalize for variation between different plates. An inter-run calibrator must have
the same sample and target between plates. If no such well exists on the PrimePCR
plate, consider designating a positive PCR control well as the IRC, and be sure that
the sample and target are labeled the same (for example, edit sample name to IRC
and target to Positive PCR).
6. After gene expression, generate analysis reports by selecting Tools > Reports… or
export the data to Microsoft Excel by selecting Export > Export All Datasheets
to Excel.
Note: PrimePCR Analysis Software is available for real-time platforms not made by Bio-Rad.
This enables easy PrimePCR plate file population with data (Cq values) generated on any
real-time PCR instrument. Visit www.bio-rad.com/PrimePCR to download the software and
instruction manual.
24 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
6 Reference Gene Selection
and Reference Gene Panels
To ensure proper normalization of loaded RNA in a gene expression experiment, use a
stable, validated reference gene(s). The reference gene(s) should maintain consistent
expression across all samples in the project regardless of treatment, source, or extraction
method. The number used and specific choice of reference genes are key factors in
determining the magnitude of change in expression that can be detected. To detect large
changes (greater than fourfold), a single reference gene may be sufficient. For smaller
changes (less than fourfold), use multiple well–validated reference genes to prevent
confounding effects produced by fluctuations in basal or uninduced expression in any single
reference gene. In addition, validate reference genes for stability using this method:
1. Choose candidate reference genes.
Select a candidate list of reference genes using published references as guidance. Use at
least five reference genes for evaluation. For your convenience, Bio-Rad offers preplated
reference gene panels using our validated and optimized PrimePCR™ assays. Each
reference gene panel contains 14 reference genes commonly used in gene expression
studies. The 96-well plate can accommodate up to six unique samples (or three samples in
duplicate or two samples in triplicate), while the 384-well plate can accommodate 24 unique
samples (or twelve samples in duplicate, eight samples in triplicate).
2. Select representative samples across the groups.
Select samples to represent all the conditions to be used in the study (for example,
treatments, tissues, time courses), ensuring that all variables within the sample groups
are evaluated.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 25
3. Isolate the RNA.
Isolate the RNA from the samples and treat with DNase using the same protocol for all
samples. Quantify and normalize the RNA to the same concentration.
4. Perform reverse transcription to cDNA.
Perform a reverse transcription reaction for each sample using the same kit, column, and
concentration. Dilute the cDNA, as needed, treating each sample the same to ensure there
are no differences from sample to sample in terms of volume and concentration from the
initial RNA input.
5. Analyze expression using real-time PCR.
Perform a real-time PCR experiment using the samples and the reference gene panel, or
your selected reference gene assays.
6. Evaluate stability.
Statistical validation of reference gene stability is detailed in a study by Vandesompele
et al. (2002) using an iterative test of pairwise variation. CFX Manager™ software includes
this method as an automated dialog. To calculate the M value for each reference gene
tested, open the data file in the Gene Expression tab and select Target Stability.
The M values will be presented in tabular format in a dialog box. The lowest M value
corresponds to the most stable expression in the tested samples, and recommended cutoff
values for M are <0.5 (for homogenous sample sets) and <1 (for heterogenous sample sets).
Reference gene stability can also be calculated using Biogazelle’s geNormPLUS or qbase+
software. Alternatively, use coefficient of variation (CV) or analysis of variance (ANOVA)
methods to evaluate stability.
Reference
Vandesompele J et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging
of multiple internal control genes. Genome Biol 3, RESEARCH0034.
26 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
7 MIQE Compliance
The minimum information for publication of quantitative real-time PCR experiments (MIQE)
guidelines allow for greater transparency in qPCR data across laboratories. These guidelines
allow scientists to investigate the quality of results presented and the ability to repeat
experiments precisely. Bio-Rad’s PrimePCR™ assays make MIQE compliance easy. Each
PrimePCR assay provides a Unique Assay ID, which can be easily tracked and reported in
publications. These IDs are provided on the website when ordering and on the specifications
sheets accompanying the product when shipping. Additionally, each PrimePCR assay has
a thorough validation report that includes which transcripts for a given gene are targeted,
and a context sequence where the amplicon can be found. For more information on how
PrimePCR assays meet MIQE guidelines, refer to bulletin 6262 (found under the Documents
tab at www.bio-rad.com/primepcr) or review the original publication (Bustin et al. 2009).
Reference
Bustin SA et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR
experiments. Clin Chem 55, 611–622.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 27
28 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
8 Frequently Asked Questions
(FAQs)
How did Bio-Rad wet-lab validate PrimePCR™ assays?
Every primer pair was experimentally tested by generating an amplification plot using
universal RNA, conducting a melt curve analysis, and calculating the efficiency and dynamic
range from a seven-point standard curve using 20–20,000,000 copies of synthetic template.
Specificity was determined by next generation sequencing of the amplicon.
Where can I find validation reports?
At www.bio-rad.com/primepcr, each assay has a PDF with validation data, including assay
information, transcripts detected, validation graphs and values, and a key for interpreting data.
Can I multiplex PrimePCR probe assays?
The fluorophores chosen for PrimePCR probes assays enable multiplex experiments;
however, not every combination of probe assays has been validated. Compare singleplex
and multiplex reactions to determine the performance of multiplex experiments. See
Multiplex Assay Considerations in Chapter 1.
Do I need to preamplify my cDNA?
Preamplification is an optional step in the PrimePCR protocol. Performing preamplification
will allow the user to perform more real-time PCR reactions from limited amounts of sample
(10 pg–100 ng cDNA).
Will Bio-Rad provide primer sequence information?
No. The MIQE context sequence of the amplicon is provided in the validation information.
Exact sequences are not provided.
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 29
What species does Bio-Rad offer?
Human and mouse for qPCR, human for ddPCR™.
Can PrimePCR products be ordered using PunchOut?
Yes.
What does guaranteed performance mean?
Assay performs well when used with Bio-Rad amplification reagents and samples of
sufficient quality.
30 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
9 Troubleshooting
Troubleshooting guide
Symptom
Cause
Treatment
Positive gDNA experimental control
gDNA in sample
Perform DNase digestion during RNA purification step
Positive NTC (no template control)
DNA contamination in workflow
Identify and eliminate source of contamination
(plastics, reagents, workspace, etc.)
Possible PCR inhibitors in reaction
Unexpectedly high Cq values and a
positive PCR experimental control value
Identify and eliminate inhibitor (see list of common
inhibitors below) by purifying samples using a postisolation cleanup kit
Wells along edge of PCR plate are
varying between technical repeats
Evaporation of PCR reactions
Seal plate better before placing in thermal cycler; use
a heat sealer for best results
Variability in reference gene Cq values Expression of reference gene is not
stable in RNA sample
Analyze expression levels of multiple reference genes
from all sample/condition combinations and choose a
stably expressed target(s)
RNA quality control assay is outside
acceptable range
RNA is degraded
Prepare and purify a new RNA sample, keeping it at
–80°C until use
Limit freeze-thaw cycles
Eliminate RNases from workspace/reagents
Spikes in early cycles of amplification
traces
Bubbles present in wells and
popping during cycling
Take care when adding samples to plates, especially
384-well plates. Position the pipet tips almost to the
bottom and dispense sample into wells, being careful
not to go to the second stop, which will expel a small
air bubble into the sample
Standard curve using synthetic
template does not appear to be linear,
or efficiency is not close to what is
described in the validation data
Loss of synthetic template during
serial dilutions
Use 10 ng/µl of tRNA in TE (pH 8.0) when diluting
template
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 31
Common PCR inhibitors.
Source of Contamination
Sample
Isolation Method
Melanin
Ethanol >1% v/v
Polysaccharides
Proteinase K
Hemoglobin
DMSO >5%
Chlorophyll
EDTA >50 mM
Polyphenolics
SDS >0.01% w/v
Heparin
Sodium acetate >5 mM
Humic acid
Mercaptoethanol
Hematin
Guanidinium
Phenol >0.2% v/v
DTT >1 mM
32 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
10Ordering Information
To order PrimePCR™ products, visit www.bio-rad.com/PrimePCR. All products must be
ordered and quoted online to ensure correct assays of interest and configuration of panels.
To check for availability of genes and determine the best panel based on your targets of
interest, visit www.bio-rad.com/primepcr_lookup and use our easy lookup tool.
PrimePCR Assays, Panels, and Plates
Catalog#Description
100-25636 —100-25641
SYBR® Green Assays
100-31225 — 100-31239
qPCR Probe Assays
100-31240 — 100-31251
ddPCR™ Probe Assays
100-25220 — 100-25225
Custom Primer Assay
100-31261 — 100-31275
Custom qPCR Probe Assay
100-31276 — 100-31281
Custom ddPCR Probe Assay
100-25031 — 100-40427
Predesigned Pathway/Disease Panels
100-25216 — 100-25219
Custom 96-Well Plates
100-25210 — 100-25212
Custom 384-Well Plates
100-25695
Reverse Transcription Control Assay
100-25694 and 100-29103
RNA Quality Assay
100-25591 and 100-29101
Positive PCR Control Assay
100-25352 and 100-25352
Genomic DNA Control Assay
100-31286
Reverse Transcription Control Probe Assay
100-31287 and 100-32858
RNA Quality Control Probe Assay
PrimePCR Assays, Panels, and Controls for Real-Time PCR | 33
Catalog#Description
100-31288
Positive PCR Control Probe Assay — FAM
100-31289
Positive PCR Control Probe Assay — HEX
100-31290 and 100-32859
Genomic DNA Control Probe Assay
100-25716
Synthetic DNA SYBR® Control Templates
100-31285
Synthetic DNA Probe Control Templates
100-41594
Custom PreAmp Assay
100-41595SYBR® Green PreAmp Assay
100-41596
Probe PreAmp Assay
Bulk Discounts
Please note that pricing and bulk discounting are subject to change.
Assay and Control Bulk Discounts
25% off 5 or more assays
Pathway and Collection Panel Bulk Discounts
25% off 5–9 plates
45% off 10–19 plates
60% off 20 or more plates
Custom Plate Bulk Discounts
Custom 96-well and 384-well plates with 1–96 unique genes:
20% off 10–19 plates
35% off 20 or more plates
Custom 384-well plates with 97 or more unique genes:
20% off 20–29 plates
35% off 30 or more plates
Related Products
To learn more about Bio-Rad’s complete solution for real-time PCR, visit
www.bio-rad.com/amplification.
34 | PrimePCR Assays, Panels, and Controls for Real-Time PCR
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 3065 7550
Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00
France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
Israel 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 1800 88 22 88 United Kingdom 020 8328 2000
Life Science
Group
10026370 Rev E
US/EG
0414
Sig 1213
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