SensiFAST™ cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The 5x TransAmp™ Buffer provides highly optimized components for efficient reverse transcription, and includes a unique blend of anchored oligo dT and random hexamer primers to ensure unbiased 3’ and 5’ coverage for enhanced data accuracy. For highly-structured RNA,increase the incubation temperature from 42C to 48 °C for 15 min

Upstream tips
The 5x TransAmp™ Buffer provides highly optimized components for efficient reverse transcription, and includes a unique blend of anchored oligo dT and random hexamer primers to ensure unbiased 3’ and 5’ coverage for enhanced data accuracy. For highly-structured RNA,increase the incubation temperature from 42C to 48 °C for 15 min

Publication protocol

RNA was isolated and checked for purity as previously described (Chu & Lidstrom, 2016). Briefly, cells were grown in the presence or absence of lanthanum and RNA was harvested using an acid phenol-chloroform extraction. We used iScript™ Reverse Transcription Supermix (Biorad) with and without reverse transcriptase to ensure all RNA was DNA-free. RNA quality and concentration was determined using Nanodrop® ND-1000. cDNA was generated using 100 ng–500 ng isolated RNA as template with the SensiFAST™ cDNA Synthesis Kit (Bioline). PCR reactions contained the following components: 400 µM primers, SensiFAST™ SYBR® No-ROX Kit (Bioline), cDNA, and ddH2O up to 10 µl volume. The PCR reactions were performed in LightCycler® Capillaries (Roche Diagnostics) and reactions were run using a LightCycler 2.0 (Roche Diagnostics). The following PCR program was used: Step 1: 95°C for 3 min, Step 2: 95°C for 10 s, Step 3: 55°C for 15 s, Step 4: 72°C for 45 s, Step 5: 72°C for 3 s (SYBR detection), Step 6: repeat steps 2 through 5 40 times. Ct values were determined using LightCycler Software Version 3.5 (Roche) and all gene expression values were normalized to 16S Ct values. All primers used for real-time quantitative reverse transcription (qRT)-PCR assays are listed in Table S1.

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Manufacturer protocol

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