10.2.8.1. High temperature reverse transcription

Using an elevated temperature for the cDNA reaction significantly reduces mis-priming events, and thus the risk of falsely detecting the incorrect strand. Common alternatives are: SuperScript-III (50oC: Peyrefitte et al., 2003; Purcell et al., 2006; Plaskon et al., 2009; Lin et al., 2009; Runckel et al., 2011), OmniScript/SensiScript (55oC: Yue and Genersch, 2005; Gisder et al., 2009), Thermoscript (60oC: Carrière et al., 2006; Horsington and Zhang, 2007) and rTth reverse transcriptase (70oC: Lanford et al., 1995; Laskus et al., 1998; Craggs et al., 2001; Celle et al., 2009). The method below is a generic one, with individual adaptations for the different reverse transcription options:

1. Mix:
1.1.  5 µl        50 ng/µl RNA,
1.2.  1 µl        10 µM tagged-cDNA primer        (0.5 µM final concentration),
1.3.  2 µl        nuclease-free water.
2. Heat 70oC for 5 min. Cool on ice for 2 min .
3.a. For SuperScript-III reactions, add:  
3.a.1.  10 µl 2x SuperScript-III buffer     (containing 1 mM dNTP),
3.a.2.  2 µl     SuperScript-III/RNAseOUT mixture,
3.a.3.  Incubate 30 min at 50oC,
3.a.4.  Inactivate 15 min at 95oC,
3.a.5.  Cool reaction to room temperature, store on ice.  
3.b. For OmniScript/SensiScript reactions, add:
3.b.1.  4 µl     5x Qiagen OneStep RT-PCR buffer,
3.b.2.  0.8 µl 10 mM dNTP           (400 µM final concentration),
3.b.3.  0.8 µl Qiagen OneStep enzyme mix,
3.b.4.  5.4 µl nuclease-free water,
3.b.5.  Incubate 30 min at 50oC. Go to section 10.2.8.4.
3.c. For ThermoScript reactions, add:
3.c.1.  4 µl     5x ThermoScript buffer,
3.c.2.  2 µl     10 mM dNTP         (1 mM final concentration),
3.c.3.  1 µl 0.1 M DTT                 (5 mM final concentration),
3.c.4 . 1 µl 40 u/µl RNAseOut,
3.c.5.  1 µl     15 u/µl         ThermoScript,
3.c.6.  4 µl     nuclease-free water,
3.c.7.  Incubate 30 min at 60oC,
3.c.8.  Inactivate 15 min at 95oC,
3.c.9.  Cool reaction to room temperature, store on ice.
3.d. For rTth reactions, add:
3.d.1.  2 µl     10x rTth buffer,
3.d.2.  0.4 µl 10 mM dNTP    (200 µM final concentration),
3.d.3.  1 µl     10 mM MnCl2  (1 mM final concentration),
3.d.4.  2 µl     2.5 u/µl rTth reverse transcriptase,
3.d.5.  6.6 µl nuclease-free water,
3.d.6.  Incubate 30 min at 70oC,
3.d.7.  Add 2 µl 10x chelating buffer (to chelate the Mn+2),
3.d.8.  Inactivate 15 min at 98oC,
3.d.9.  Cool reaction to room temperature, store on ice.