iScript cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Reverse transcribe for 20 min at 46C	For downstream PCR use only 1/10 of the reaction volume (typically 2ul)

Protocol tips
Reverse transcribe for 20 min at 46C
Downstream tips
For downstream PCR use only 1/10 of the reaction volume (typically 2ul)

Publication protocol

Total RNA was extracted from mouse aortas using an RNeasy Mini Kit (Qiagen, Valencia, CA). The
isolated RNA was treated with DNase I to remove genomic DNA contamination. RNA quality was
verified by determining the ratio of the absorbance at 260
and 280 nm. The A260/280 ratio of the isolated RNA was 1.8-2.0. Reverse transcription was performed
with 1 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad), according to the manufacturer's
protocol (1;2). PCR primers used for amplification of mouse genes were as follows: MCP-1, forward 5’-
CCTGGATCGGAACCAAATGA-3’, reverse 5’-CGGGTCAACTTCACATTCAAAG-3’; VCAM-1,
forward 5’-TTTTCCAATATCCTCAATGACGGG-3’, reverse 5’-
CCTCACTTGCAGCACTACGGGCT-3’; IFN-γ, forward 5’-AACGCTACACACTGCATCTTGG-3’,
reverse 5’-GACTTCAAAGAGTCTGAGG-3’; iNOS, forward 5’-CAGAAGCAGAATGTGACCATC3’, reverse 5’-CTTCTGGTCGATGTCATGA-3’; Cox-2, forward 5’-
GCAAATCCTTGCTGTTCCAACCCA-3', reverse 5’-TTGGGGATCCGGGATGAACTCTCT-3’;
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5’-
GCAGACGGTGCAGCGCATCTTGG-3′, reverse 5’-TGGGTACGTATACGGCTTGTCAC-3’. The
PCR primers were designed using http://www.ncbi.nlm.gov/tools/primer-blast. The primers should
specifically span an intron of target genes, to ensure that only cDNA and not genomic DNA was
amplified. To validate the primers, we ran the PCR products on agarose gel, which revealed single bands
of the correct size. To determine the efficiency of the primers, we ran standard curves. The slopes of the
standard curves (Ct vs. concentration) were close to -3.32. GAPDH was used as a reference gene
because it is a popular housekeeping standard used in gene expression studies (3).
PCR Reactions were performed in a CFX96 Real-Time System (Bio-Rad). PCR reaction mixtures
(20 µl) contained 8 µl of ddH2O, 10 µl of 1× iQTM SYBR Green SuperMix (Bio-Rad), 1 µl of each
specific primer (250 nM), and 1 µl of first-strand cDNA template (40 ng). The quantitative PCR
program included an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at
95°C for 15 s, annealing for 30 s at 60°C, and extension for 30 s at 72°C. For melting curve analysis, a
dissociation step cycle (65°C for 10 s, and then 0.5°C for 10 s until 95°C) was added. The reactions
were set up in 96-well format Microseal PCR plates (Bio-Rad) in triplicate. The existence of single
peaks in melting curve analysis was used to confirm gene-specific amplification and rule out nonspecific amplification and primer-dimer generation. All experiments were performed in triplicate. The
relative RNA amount was calculated with the 2-ΔΔCt method and normalized with internal control
GADPH as described by Pfaffl (4).

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Manufacturer protocol

Download the product protocol from Bio-Rad Laboratories for iScript cDNA Synthesis Kit below.

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