“…This lack of strand specificity has been attributed to a combination of factors, including self-priming of the RNA due to secondary hairpin structures (Haddad et al, 2006;Lanford et al, 1994;Lerat et al, 1996;Stahlberg et al, 2004;Timofeeva and Skrypina, 2001;Tuiskunen et al, 2010), false priming of the incorrect strand (Gunji et al, 1994;Lanford et al, 1994;Lin et al, 2002;Sangar and Carroll, 1998) and random priming by contaminating endogenous or exogenous nucleic acids (Gunji et al, 1994;Piche and Schernthaner, 2003;Timofeeva and Skrypina, 2001). Attempts to overcome these problems include performing RT reactions at high temperatures (Haddad et al, 2007;Komurian-Pradel et al, 2004), use of the thermostable RTth enzyme (de Paula et al, 2009;Lanford et al, 1994;Radkowski et al, 2002;Selva et al, 2004), use of tagged RT primers (Peyrefitte et al, 2003;Plaskon et al, 2009;Purcell et al, 2006;Vashist et al, 2012) or a combination of tagged primers and RTth enzyme (Craggs et al, 2001). It was shown that the approach employing tagged primers in combination with high RT temperature greatly improved the specificity of the RT reactions.…”