RNA LABELING METHOD
A method of sample analysis is provided. In certain embodiments, the method involves: a) obtaining a fragmented RNA sample comprising fragments of long RNA molecules and short RNA molecules; b) ligating an adaptor to an end of the RNA of the fragmented RNA sample to produce an adaptor-ligated sample; c) hybridizing said adaptor-ligated sample to an array of nucleic acid probes; and d) reading said array to obtain an estimate of the abundance of a long RNA in the RNA sample and an estimate of the abundance a small RNA in the RNA sample.
1 . A method of sample analysis comprising:
a) obtaining a fragmented RNA sample comprising:
i. fragments of long RNA molecules, wherein the long RNA molecules are at least 200 nucleotides in length; and
ii. short RNA molecules of less then 200 nucleotides in length;
b) ligating an adaptor to an end of the RNA of said fragmented RNA sample to produce an adaptor-ligated sample;
c) hybridizing said adaptor-ligated sample to an array of nucleic acid probes; and
d) reading said array to obtain an estimate of the abundance of a fragmented long RNA in said RNA sample and an estimate of the abundance of a small RNA in said RNA sample.
2 . The method of claim 1 , wherein said fragmented RNA sample is made by exposing an initial RNA sample comprising intact long RNA and short RNA to fragmentation conditions.
3 . The method of claim 2 , wherein said exposing comprises contacting said initial RNA sample with a divalent cation at a temperature of at least 50° C.
4 . The method of claim 2 , wherein said initial RNA sample comprises total cellular RNA.
5 . The method of claim 2 , wherein said initial RNA sample comprises total cellular RNA from which tRNA and rRNA has been removed.
6 . The method of claim 1 , wherein said fragmented RNA sample is obtained from formalin-fixed paraffin embedded tissue (FPET) sample.
7 . The method of claim 1 , wherein said short RNA molecules comprise small RNA molecules selected from the group consisting of short interfering RNA (siRNA) molecules, microRNA (miRNA) molecules, tiny non-coding RNA (tncRNA) molecules or small modulatory RNA (smRNA) molecules.
8 . The method of claim 1 , wherein said long RNA molecules comprise lincRNA molecules or mRNA molecules.
9 . The method of claim 1 , wherein said method comprises ligating an adaptor to the 3′ end of the RNA of said fragmented RNA sample.
10 . The method of claim 1 , wherein said method comprises ligating an adaptor to the 5′ end of the RNA of said fragmented RNA sample.
11 . The method of claim 1 , wherein the RNA of said fragmented RNA sample is labeled prior to or after said ligating step b).
12 . The method of claim 1 , wherein said adaptor is a labeled adaptor.
13 . The method of claim 1 , wherein said adaptor is in the range of 6 to 12 nucleotides in length.
14 . The method of claim 1 , further comprising treating said fragmented RNA sample with a polynucleotide kinase or a phosphatase in the absence of ATP prior to said ligating step b).
15 . The method of claim 1 , wherein the adaptor-ligated sample comprises
i. adaptor-ligated short RNA comprising an adaptor portion and a short RNA portion; and
ii. adaptor-ligated long RNA fragments comprising an adaptor portion and a long RNA portion;
and wherein said array comprises
i. a first nucleic acid probe that comprises a nucleotide sequence that is complementary to both said adaptor portion and said short RNA portion of said adaptor-ligated short RNA; and
ii. a second probe that comprises a nucleotide sequence that is complementary to the long RNA portion of said adaptor-ligated long RNA but not said adaptor portion of said long RNA.
16 . An array comprising:
i. a plurality of first probes that comprise nucleotide sequences that are complementary to different adaptor-ligated short RNAs, wherein said first probes are complementary to both the adaptor portions and the short RNA portions of said adaptor-ligated short RNAs; and
ii. a plurality of second probes that comprise nucleotide sequences that are complementary to different long RNAs.
17 . The array of claim 16 , wherein said adaptor is in the range of 6 to 12 nucleotides in length.
18 . The array of claim 16 , wherein said array comprises at least 100 of said first probes and at least 1000 of the second probes.
19 . A kit comprising:
a) an array of claim 16 ;
b) an RNA ligase; and
c) an adaptor.
20 . The kit of claim 19 , further comprising instructions for performing the method of claim 1 .