Total RNA (10 ng) from different blood samples was amplified for 2 or 8 hours using the QuantiTect Whole Transcriptome Kit. cDNA yields were determined using PicoGreen reagent.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit. This was followed by real-time PCR using 10 ng cDNA, primers specific for β-actin, and the QuantiTect SYBR® Green PCR Kit. Amplicons corresponding to the 5' and 3' regions of the β-actin transcript were detected with similar CT values, indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions.
cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.
Replicate RNA samples (10 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR of the indicated targets was then performed using 10 ng cDNA and the QuantiFast Probe PCR Kit on the Mx3005P system. The overlapping curves indicate highly reproducible whole transcriptome amplification. ACTB: β-actin; HPRT1: Hypoxanthine phosphoribosyltransferase 1.
cDNA is first synthesized from template RNA and then ligated (not shown). REPLI-g DNA polymerase moves along the cDNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, allowing high yields of cDNA to be generated.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit for 2 hours (WTA 2 h) or 8 hours (WTA 8 h), followed by real-time PCR of 5 targets using 10 ng cDNA. As a control, RNA was reverse transcribed without amplification using the Sensiscript RT Kit (Non-WTA), and 1/5 of the RT reaction was used in real-time PCR. Real-time PCR was carried out using the QuantiTect Probe PCR Kit. [A] Relative gene expression levels of 5 transcripts (normalization to HPRT1) are similar in the WTA 2 h, WTA 8 h, and Non-WTA samples, as shown by the overlapping points. [B] A plot of ΔCT values (CT value for each target minus CT value for internal reference β-actin) for WTA 8 h samples (Y-axis) against those for Non-WTA samples (X-axis) is highly linear. The data in [A] and [B] demonstrate that the QuantiTect Whole Transcriptome Kit preserves the transcript profile. TP53: Tumor protein p53; NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; HPRT1: hypoxanthine phosphoribosyltransferase 1; TBP: TATA box binding protein; TNFRSF6B: tumor necrosis factor receptor superfamily, member 6b, decoy.