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reverse transcription

1. Prepare the following reaction mixture on ice.
   Prepare a slightly larger amount of master mix than is required to compensate for pipetting losses. After dispensing aliquots of this mix into the micro tubes, add the RNA sample.
   
   [For 1 reaction]
   

   Reagent	Amount	Final concentration
   5 × PrimeScript™ Buffer (for Real Time)	2 µl	1 ×
   PrimeScript™ RT Enzyme Mix I	0.5 µl
   Oligo dT Primer (50 µM)*1	0.5 µl	25 pmol
   Random 6 mers (100 µM)*1	0.5 µl	50 pmol
   total RNA
   RNase Free dH2O
   Total	10 µl*2

   
   

   *1.	Using both Oligo dT Primer and Random 6 mers, efficient synthesis of cDNA from total mRNA can be accomplished. The required amount of primer for exclusive use of each primer or a Gene Specific primer is as follows.

   
   

   Primer	Amount	Total Amount (pmol)
   Oligo dT Primers (50 µM)	0.5 µl	25 pmol
   Random 6 mers (100 µM)	0.5 µl	50 pmol
   Gene Specific Primer (2 µM)	0.5 µl	1 pmol

   
   

   *2.	It is possible to scale up the RT reaction as needed. Up to 500 ng of total RNA can be reverse transcribed in 10 µl of the reaction mixture.

   
   
2. Incubate the reaction mixture under the following condition.
   37°C , 15 minutes^*3 (Reverse transcription)
   85°C , 5 sec (Inactivation of reverse transcriptase with heat treatment)
   4°C
   
   

   *3.	When using a Gene Specific Primer: Perform the reverse transcription at 42°C for 15 minutes. If non-specific amplification products are observed at the PCR step, resetting this temperature to 50°C may improve the results.

   
   

   Note:

   The volume of the reaction mixture obtained in step 2 must be less than 10% of the total PCR reaction volume for real-time PCR.