Using Qubit to quantify microRNAs for TruSeq in small RNA sequencing

[candicechu]

Hi all,

For small RNA sequencing, we are requested to submit either

(1) 1 ug of total RNA

or

(2) 10-15 ng of microRNA

Does anyone have the experience using Qubit RNA HS for total RNA quantification or Qubit microRNA assay for microRNA quantification?

Thanks!

[nucacidhunter]

miRNA assay would be more specific. Alternatively, samples can be run on Small RNA Chip which will give an indication of miRNA isolation success and concentration.

[candicechu]

miRNA assay would be more specific. Alternatively, samples can be run on Small RNA Chip which will give an indication of miRNA isolation success and concentration.

Thanks nucaidhunter! I consulted the tech support of Illumina before and they told to observe the appearance of miRNA peak in small RNA chip but not to trust the concentration given by bioanalyzer.

[nucacidhunter]

Illumina’s comment regarding small RNA Chip quantification accuracy is correct. However, if you submit them to a core facility for library prep they most likely will use small RNA Chip for both purposes to avoid wasting sample. Accuracy of Qubit quantification also can vary widely depending on volume of sample used for quantification and other factors. Input quantity for the small RNA library prep has a relatively wide window so inaccuracy in input (+/-2x) is well tolerated.