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Determining the optimal reference gene – Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix User Manual

Page 11

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SsoAdvanced

Universal SYBR

®

Green Supermix Instruction Manual

| 5

Real-Time PCR Validation for Gene Expression Experiments

The following validation experiments are critical for obtaining valid and publishable real-time
PCR data following the MIQE guidelines. These simple-to-follow experiments should be
completed prior to starting a new real-time PCR project.

Determining the Optimal Reference Gene

To properly perform a gene expression experiment, it is imperative that an optimal
reference gene(s) is used. The reference gene(s) must maintain a consistent expression
level across all samples in the project regardless of treatment, source, or extraction
method. The variation in reference gene expression is somewhat dependent on the level
of fold change discrimination desired. For example, if a twofold change in expression is
important, then the reference gene should have little to no variation in expression. However,
if a 20-fold change in expression is important, then the reference gene expression can have
some variability. To validate a reference gene(s), follow these steps:

1. Begin searching for a candidate list of reference genes by searching publications, speaking

with researchers using similar model systems, and mining microarray data, if available.
Minimally, five reference genes should be selected for evaluation. For your convenience,
Bio-Rad offers pre-plated reference gene panels using our highly validated and optimized

PrimePCR assays

.

2. From your experiment, randomly select a few samples from each group (for example,

treatments, time courses, sources) ensuring that you evaluate all variable sample groups.

3. Isolate the RNA and DNase-treat using the same protocol for all samples. Quantify and

normalize the RNA to the same concentration.

Table 2. Thermal cycling protocol.

Amplification

Polymerase

Annealing/

Activation

Extension +

Setting/

and DNA

Denaturation Plate Read

Melt-Curve

Real-Time PCR System

Mode

Denaturation at 95/98°C

at 60°C** Cycles Analysis

Bio-Rad

®

CFX96

,

CFX384

, CFX96 Touch

,

SYBR

®

only

10–30 sec

CFX384 Touch

,

CFX Connect

Bio-Rad

®

iQ

5, MiniOpticon

, Standard

15–30 sec

Chromo4

, MyiQ

ABI 7500, StepOne,

Fast

5–15 sec

10–30 sec 35–40

StepOnePlus, 7900HT

Standard

60 sec

and ViiA7

Roche LightCycler 480

Fast

10–30 sec

Standard

60 sec

Qiagen Rotor-Gene and

Fast

10–30 sec

Stratagene Mx series

* 98°C is highly recommended for genomic DNA template to ensure complete denaturation.

** Shorter annealing/extension times (1–10 sec) can be used for amplicons <100 bp. Longer annealing/extension

times (30–60 sec) can be used for amplicons >250 bp, GC- or AT- rich targets, crude samples, or for higher input
amounts (for example, 100 ng of cDNA or 50 ng of genomic DNA).

30 sec at
95°C for
cDNA
or

5–15 sec

2–3 min at
98°C for
genomic
DNA*

65–95°C

0.5°C

increment

2–5

35–40 sec/step

(or use

instrument

default

setting)