Hi 

I guess you extracted small RNA from total RNA from your samples. since small RNA are subject to degradation as total RNA, do you have an idea on the quality from your samples? if not, just try to test on agilent bioanalyzer or simply agarose gel.

fred

Aleksej,

Do you have nanodrop or something similar to analyze the quantity and quality of your RNA? If so, it is worth to check it. Additionally, are you adding an inhibitor of RNases when preparing the RT reaction mix, such as RNAsin or RNAse out? Did you heat first the RNA with primers and dNTPs at 70 celsius for 5 min before adding the RT enzyme, RNases inhibitor and buffer for the RT enzyme? Those are key steps to always obtain good cDNA.

Hi Aleksej,

a thermostable reverse transcriptase has recently become available that is intended for structured RNA applications. It has been successfully used with tRNAs. A few example publications below:

Zheng G, Qin Y, Clark WC, Dai Q, Yi C, He C, Lambowitz AM, Pan T. Efficient and quantitative high-throughput tRNA sequencing. Nat Methods. 2015;12 (9) :835-7.

Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S, et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA [Internet]. 2013;19 (7) :958-70.

Hi Alexej,

You may find answers on some of your questions in Wittig , B., Wittig, C. (1978) Nucleic Acids Res. 5(4): 1165-78; and Motorin, Y. et al. (2007) Methods in Enzymology 425: 21-52.