The size of the fragments will depend on 3 things (genomic DNA contamination excluded):
1- The quality of your RNA
Higher the quality, higher the fragments size.
2- The mRNA content of your sample
If you have a extremely good quality RNA containing Titin mRNA, you will have the longest possible cDNA strand.
3- The primers you use for cDNA synthesis.
a) Hexamers (convert any RNA strand to cDNA, even degraded fragments)
b) Olido-dT (only mRNA is converted to cDNA, mRNA length determines the length of cDNA. Titin wins again here)
c) Target specific primers (usually for cloning purposes, this is the best way you can control the fragment size).
The size of the fragments will depend on 3 things (genomic DNA contamination excluded):
1- The quality of your RNA
Higher the quality, higher the fragments size.
2- The mRNA content of your sample
If you have a extremely good quality RNA containing Titin mRNA, you will have the longest possible cDNA strand.
3- The primers you use for cDNA synthesis.
a) Hexamers (convert any RNA strand to cDNA, even degraded fragments)
b) Olido-dT (only mRNA is converted to cDNA, mRNA length determines the length of cDNA. Titin wins again here)
c) Target specific primers (usually for cloning purposes, this is the best way you can control the fragment size).
with our single cell libraries (oligo dT-based), size is mostly determined by the RT you are using: Superscript II (Invitrogen) and Primescript (Takara) are the best here, giving libraries with approximately 2 kb average size,
I agree with sir Korcan Ayata I think beside this The Enzyme Reverse Transcriptase ... it should be of high fidelity...
Thanks everybody!!
can you suggest me a protocol to follow to understand how to get the size I want?
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