Comment on my cDNA lib electropherograms - new to this

[PhDstudent]

Hi there,

Just starting to make cDNA libraries and not sure how the electropherogram should look when I do fragment size analysis with Bioanalyzer High sensitivity DNA chip.

So I used the KAPA Stranded mRNA-seq kit. Fragmentation is thermal, selecting for 200-300bp fragments.

Goal is DE between drug treatments compared to control. Right now just troubleshooting to make sure our library prep is up to par before doing the real libraries.

Sample 2: 2ug total eukaryotic RNA from mouse primary neuron culture with 10x PCR cycles
Sample 4: 1ug same type of RNA with 10x PCR cycles
Sample 5: 1ug same type of RNA with 8x PCR cycles

My feeling is that Samples 2 & 4 are just over amplified/too much cDNA loaded for the high-sensitivity DNA chip.

Please give me feedback on my electropherograms, would be very greatful for any positive or negative feedback.

Cheers.

[pmiguel]

Sounds reasonable. If your PCR reaction becomes depleted for reactants, then you end up with "bubble products". That is, the ends of the amplicons are all basically the same sequences, so they can anneal even if their inserts don't match. So after denaturation if no product strand is created, then the single-stranded amplicon will likely run into another single-stranded amplicons and their ends with hybridize. These "bubble products" run strange on bioanalyzer chips -- aberrantly large for their molecular weight.

--
Phillip

[PhDstudent]

Thanks for the response! Do you find though that the downstroke of the curve should be more abrupt similar to the upstroke of the curve?

[Jennifer Pavlica]

Dear PhDstudent,

I am a Sr. Support and Applications Scientist with Kapa Biosystems. After reviewing your BioAnalyzer traces, it appears that your libraries may be slightly overamplified, most noticeably in Sample 2. With future samples, I recommend decreasing the amplification by a cycle or two.

However, it is probable that the root cause of the bimodal peaks observed in Samples 2 and 4 are a result of sample overloading on the High Sensitivity chip. Increasing the dilution factor prior to assessment on the BioAnalyzer should alleviate this issue.

I hope you find this information useful.

Best,

Jennifer Pavlica