Reverse Transcriptase (RT)

The first step in RT-qPCR uses a Reverse Transcriptase to generate the complementary DNA (cDNA) of your RNA sample. Depending on your application, you may use random or gene specific primers.

Review available kits and consider which Reverse Transcriptase to use for your target application. The first protocol is for using MultiScribe Reverse Transcriptase with random primers for RT-qPCR. We use random primers to non-specifically generate cDNA for all RNAs in the sample, which will include rRNA, mRNA and ncRNAs. In E. coli we use the 16S rRNA as an endogenous control (known to be expressed at a relatively constant level) so that if samples have variable loading in the end, all relative mRNA levels can be normalized and compared. The second protocol (below) is for SuperScript IV Reverse Transcriptase. SuperScript has been used for selective RT with gene-specific primers for various RNA-seq projects.

Important Principles to Remember

1. Use RNase free pipettes, tips, etc for setting up the reaction.

2. Maintain RNase free environment using RNase away and disposable sleeves and lab coat.

3. Perform the reaction setup in a clean hood that was not used for the RNA extraction (optional).

MultiScribe Reverse Transcriptase

When in doubt, review the manufactures notes and protocols here.

1. Add 180 µL DEPC-treated water to each sample to bring the concentration to 10 ng/µL Total RNA and 25 ng/µL Yeast tRNA.

2. Prepare RT master mix on ice (RT Kit: Applied Biosystems/ThermoFisher4368814).

Component	For 15 µL Sample	Total Volume for 18 Reactions (example)	For 22.5 µL Sample	Total Volume for 17 Reactions (example)
10x RT Buffer	3 µL	54 µL	4.5 µL	76.5 µL
25x dNTP Mix	1.2 µL	21.6 µL	1.8 µL	30.6 µL
10x RT Random Primers	3 µL	54 µL	4.5 µL	76.5 µL
Nuclease Free Water	6.3 µL	113.4 µL	9.45 µL	160.65 µL
Multiscribe Reverse Transcriptase*	1.5 µL	25.5 µL	2.25 µL	36 µL

*Before adding remove 13.5 uL/20.25 uL for a control.

1. Pipet 15 uL/22.5 uL of master-mix into each PCR tube for each reaction.

2. Pipet 15 uL/22.5 uL of diluted RNA into each PCR tube.

3. Spin down tubes.

4. Put PCR tubes into Thermocycler and run the following procedure. Set thermocycler to respective reaction volumes.

	Step 1	Step 2	Step 3	Step 4
Temperature (C)	25	37	85	4
Time (Minutes)	10	120	5	Infinity

cDNA can be left in the reaction tubes and stored in the 4°C fridge for a few days, or in the -20°C freezer up to a couple of months.

SuperScript IV Reverse Transcriptase

*Daniel insert protocol here