I have been preparing NGS Library, where the samples input volumes, conditions followed and the PCR Cycles are same but still the concentration obtained was uneven and the fragments size where also differ from sample to sample. What could be the possible reason for this uneven results.
Hari Shankar Gadri, Yes sir. mRNA Library: The sample concentration was 12.6ng/ul. The input sample volume taken for lib. prep. was 40uL (i.e: 504ng). The kit I used recommends RNA input from 10ng - 1ug.
Still, in the end, I got a Library conc. as 0.24 ng/uL (1.22 nM/uL).
Fragment size ~350bp.
Kavya M N, Mam, Can you give me suggestions on which particular step during size selection or adapter cleanup we have to be careful?
What I did during size selection was for RNA fragment size ~350 bp and library Fragment Size ~450bp. I used 2 rounds of bead selection (1st - 30uL & 2nd - 20uL), followed by 80% EtOH wash and eluted in Low EDTA TE and proceeded further.
I think your sample concentration is very low. you will amplify your samples to increase concentration. But sometimes the product overamplifies that case in our result bias and artifact.
But I strongly suggest you again isolate your product.
Vishali S P R How do you measure mRNA concentration? We're struggling at this step, Qubit seems not to be working for our samples.
Vilte Cereskaite We do not specifically measure mRNA concentration; instead, we quantify total RNA using Qubit 4 and also assess RIN to ensure the quality of RNA.
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