Hello everyone,
I am new to the RNA 3' Sequencing experiment using a protocol developed in house based on Illumina TruSeq mRNA sample prep. The library, however, did not get amplified after many tries. So I started to impose quality control steps and see what went wrong.
The starting material is 6ug human total RNA, extracted 100-250ng mRNA. The mRNAs were then fragmented in 85C for 10 mins in the 5x First Stranded buffer from Superscript III kit. I took 2uL fragmented mRNA to run a Bioanalyzer RNA pico assay, which showed a peak around the lower range between 200-500 bp. The samples were then undergone cDNA synthesis (clean up by Qiagen kit), 3'A tailing (clean up by Qiagen kit), linker ligation (clean up by Ampure beads) and PCR (clean up by Ampure beads).
While mRNA fragmentation seemed to have acceptable pattern, all of a sudden there was a peak around 500-700 bp in cDNA and 3'A tailing product, but the the peak disappear in the linker ligation product. And even more strangely, most products from PCR was around 200-300 bp, back to expected. If you refer to the attached figure you know what I mean.
I discussed with my colleague who has experience in RNA Seq, and she said this was totally weird. Has anyone here ever looked at the mRNA and cDNA profiles in these intermediate steps? What are they supposed to look like? And what could have caused this strange phenomenon here? Desperate here! Any thoughts will be much appreciated!!!
I am new to the RNA 3' Sequencing experiment using a protocol developed in house based on Illumina TruSeq mRNA sample prep. The library, however, did not get amplified after many tries. So I started to impose quality control steps and see what went wrong.
The starting material is 6ug human total RNA, extracted 100-250ng mRNA. The mRNAs were then fragmented in 85C for 10 mins in the 5x First Stranded buffer from Superscript III kit. I took 2uL fragmented mRNA to run a Bioanalyzer RNA pico assay, which showed a peak around the lower range between 200-500 bp. The samples were then undergone cDNA synthesis (clean up by Qiagen kit), 3'A tailing (clean up by Qiagen kit), linker ligation (clean up by Ampure beads) and PCR (clean up by Ampure beads).
While mRNA fragmentation seemed to have acceptable pattern, all of a sudden there was a peak around 500-700 bp in cDNA and 3'A tailing product, but the the peak disappear in the linker ligation product. And even more strangely, most products from PCR was around 200-300 bp, back to expected. If you refer to the attached figure you know what I mean.
I discussed with my colleague who has experience in RNA Seq, and she said this was totally weird. Has anyone here ever looked at the mRNA and cDNA profiles in these intermediate steps? What are they supposed to look like? And what could have caused this strange phenomenon here? Desperate here! Any thoughts will be much appreciated!!!
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