Induro^® Reverse Transcriptase Protocol (NEB #M0681)

First Strand cDNA Synthesis (Standard Protocol)

If denaturation of template RNA is desired, use the following protocol. Thaw components on ice and mix by inverting several times.

1. Mix RNA sample and primer d(T)₂₃VN in a sterile RNase-free microfuge tube.
    

   COMPONENT	VOLUME
   Total RNA	up to 1 µg*
   d(T)23VN (50 µM) [NEB #S1327] or Random Primer Mix (60 µM) [NEB #S1330]	2 µl
   10 mM dNTP [NEB #N0447]	1 µl
   Nuclease-free H2O	To a total volume of 10 µl

    * 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA

    
2. Denature sample RNA/primer for 5 minutes at 65°C. Spin briefly and put promptly on ice.
    
3. Add the following components to the same tube (to a total volume of 20 µl.)
    

   COMPONENT	VOLUME
   5X Induro RT Reaction Buffer	4 µl
   RNase Inhibitor, Murine (40 U/µl) [NEB #M0314]	0.2 µl
   Induro Reverse Transcriptase (200 U/µl)	1 µl
   Nuclease-free H2O	4.8 µl

   
    
4. Incubate the 20 μl cDNA synthesis reaction at the below conditions. If Random Primer Mix is used, an incubation step at 25°C for 2 minutes is recommended before the 55°C incubation.
    

   INCUBATION STEPS	TIME
   First strand cDNA Synthesis	10 min @ 55°C**
   Inactivation	1 min @ 95°C

   ** Induro Reverse Transcriptase can be used at 50–60°C. A longer incubation time up to 30 minutes may increase the yield of long cDNA products.

    
5. The cDNA product should be stored at -20°C.  For downstream PCR application, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
    

First Strand cDNA Synthesis (Quick Protocol)

Thaw components on ice and mix by inverting several times.

1. Mix the following components in a sterile RNase-free microfuge tube.
    

COMPONENT	VOLUME
Total RNA	up to 1 µg*
d(T)23VN (50 µM) [NEB #S1327] or Random Primer Mix (60 µM) [NEB #S1330]	2 µl
10 mM dNTP [NEB #N0447]	1 µl
RNase Inhibitor, Murine (40 U/µl) [NEB #M0314]	0.2 µl
5X Induro RT Reaction Buffer	4 µl
Induro Reverse Transcriptase (200 U/µl)	1 µl
Nuclease-free H2O	To a total volume of 20 µl

* 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA


 
2. Incubate the 20 µl cDNA synthesis reaction at the below conditions. If Random Primer Mix (NEB # S1330) is used, an incubation step at 25°C for 2 minutes is recommended before the 55°C incubation.

   INCUBATION STEPS	TIME
   First strand cDNA Synthesis	10 min @ 55°C**
   Inactivation	1 min @ 95°C

   ** Induro Reverse Transcriptase can be used at 50–60°C. A longer incubation time up to 30 minutes may increase the yield of long cDNA products.

   
    
3. The cDNA product should be stored at -20°C.  For downstream PCR application, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
    

First Strand cDNA Synthesis (No-RT Negative Control Reaction)

Thaw components on ice and mix by inverting several times.

1. Mix the following components and incubate at 55°C for 10 minutes, followed by 95°C for 1 minute.
    

   COMPONENT	VOLUME
   Total RNA	up to 1 µg*
   d(T)23VN (50 µM) [NEB #S1327] or Random Primer Mix (60 µM) [NEB #S1330]	2 µl
   10 mM dNTP [NEB #N0447]	1 µl
   RNase Inhibitor, Murine (40 U/µl) [NEB #M0314]	0.2 µl
   5X Induro RT Reaction Buffer	4 µl
   Nuclease-free H2O	To a total volume of 20 µl

   * 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA