Methods for RNA quantification
Provided herein are methods, compositions, systems, devices, and kits for quantification of RNA, and determination of efficiency of reverse transcription of RNA to cDNA.
1. A method for determining a quality of RNA of an RNA sample, comprising:
hybridizing to RNA of an RNA sample a plurality of oligonucleotides comprising a target specific region that specifically binds to a target in an mRNA molecule of one or more reference genes in the RNA sample, wherein each of the plurality of oligonucleotides comprises a stochastic barcode comprising a molecular label, wherein at least 10% of the plurality of oligonucleotides comprises different molecular labels, and wherein the one or more reference genes are endogenous to the RNA sample;
extending the hybridized plurality of oligonucleotides to generate a plurality of nucleic acid molecules of the one or more reference genes;
counting the number of molecular labels associated with the plurality of nucleic acid molecules;
determining if an amount of RNA that can be converted to cDNA in the RNA sample is sufficient for a downstream application, wherein the amount of RNA that can be converted to cDNA is based on the number of molecular labels associated with the one or more reference genes, wherein the determining comprises:
comparing the number of molecular labels associated with the one or more reference genes to:
a reference value for the number of molecular labels associated with the one or more reference genes; or
the total amount of RNA in the RNA sample; and
performing the downstream application utilizing the RNA of the RNA sample when the determined amount of RNA that can be converted to cDNA in the RNA sample is sufficient for the downstream application, or not performing the downstream application utilizing the RNA of the RNA sample when the amount of RNA that can be converted to cDNA in the RNA sample is not sufficient for the downstream application.
2. The method of claim 1 , wherein at least one of the one or more reference genes is a housekeeping gene.
3. The method of claim 1 , wherein the RNA sample comprises total RNA.
4. The method of claim 1 , wherein the RNA sample comprises less than 2 ng of RNA.
5. The method of claim 1 , wherein the extending step comprises reverse transcription of the mRNA molecule hybridized with an oligonucleotide of the plurality of oligonucleotides.
6. The method of claim 1 , wherein each of the plurality of oligonucleotides comprises a binding site for a primer, and comprising amplifying the plurality of nucleic acid molecules using a primer that binds to the binding site.
7. The method of claim 6 , wherein each of the plurality of oligonucleotides or the primer comprises an optically detectable label.
8. The method of claim 7 , wherein the optically detectable label is a fluorescent label.
9. The method of claim 7 , wherein the plurality of oligonucleotides comprises different fluorescent labels for each of the one or more reference genes.
10. The method of claim 1 , wherein counting the number of molecular labels associated with the plurality of nucleic acid molecules comprises determining the sequences of the plurality of nucleic acid molecules by sequencing.
11. The method of claim 1 , wherein counting the number of molecular labels associated with the plurality of nucleic acid molecules comprises hybridizing the plurality of nucleic acid molecules to a microarray comprising a plurality of probes that specifically binds to the molecular labels of the plurality of oligonucleotides.
12. The method of claim 1 , further comprising:
adding a plurality of spike-in RNA molecules to the RNA sample, wherein each of the plurality of spike-in RNA molecules comprises a stochastic barcode comprising a molecular label, wherein at least 10% of the plurality of spike-in RNA molecules comprises different molecular labels, and wherein the plurality of spike-in RNA molecules comprises a known amount of associated molecular labels;
hybridizing a reverse-transcription oligonucleotide to the plurality of spike-in RNA molecules;
extending the reverse-transcription oligonucleotide to generate a second plurality of nucleic acid molecules;
counting the number of molecular labels associated with the second plurality of nucleic acid molecules; and
determining the efficiency of reverse-transcription based on the ratio of the number of molecular labels associated with the second plurality of nucleic acid molecules to the known amount of molecular labels associated with the plurality of spike-in RNA molecules,
wherein determining if an amount of RNA that can be converted to cDNA in the RNA sample is sufficient for a downstream application further comprises comparing the amount of RNA that can be converted to cDNA to the determined efficiency of reverse-transcription.
13. The method of claim 12 , wherein counting the number of molecular labels associated with the second plurality of nucleic acid molecules comprises determining the sequences of the second plurality of nucleic acid molecules by sequencing.
14. The method of claim 12 , wherein counting the number of molecular labels associated with the second plurality of nucleic acid molecules comprises hybridizing the second plurality of nucleic acid molecules to a microarray.
15. The method of claim 12 , wherein comparing the amount of RNA that can be converted to cDNA to the determined efficiency of reverse-transcription comprises normalizing the amount of RNA that can be converted to cDNA against the efficiency of reverse-transcription.
16. The method of claim 1 , wherein the RNA sample comprises an RT inhibitor.
17. The method of claim 1 , wherein the RNA sample comprises chemically modified RNA.
18. The method of claim 1 , wherein the RNA sample is an mRNA sample, and wherein more than 70% of RNA molecules in the RNA sample have a length of less than 1000 nt.
19. The method of claim 1 , wherein the downstream application comprises one or more of qPCR, microarray and sequencing.