Transcriptome In Vivo Analysis (TIVA) and Transcriptome In Situ Analysis (TISA)

A method of detecting a nucleic acid molecule from a cell, the method comprising the steps of:

• introducing a hybrid nucleic acid molecule into the cell, the hybrid nucleic acid molecule comprising a first, second, and third oligonucleotide, wherein the first oligonucleotide is linked to the second oligonucleotide through a first photocleavable linker and the second oligonucleotide is linked to the third oligonucleotide through a second photocleavable linker, wherein the first oligonucleotide comprises one or more terminal deoxynucleotides and an antisense oligonucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid molecule of the cell;
• activating the hybrid nucleic acid molecule under conditions suitable for binding of the first oligonucleotide to the target nucleic acid molecule of the cell, wherein the activation cleaves the photocleavable linkers in the molecule;
• allowing the formation of a complex comprising the target nucleic acid molecule hybridized to the first oligonucleotide;
• and
• synthesizing cDNA from the target nucleic acid molecule, using the first oligonucleotide and the one or more terminal deoxynucleotides as a primer, wherein the target nucleic acid molecule comprises RNA,
• wherein the one or more terminal deoxynucleotides comprises one or more thymidines, wherein the first oligonucleotide comprises 2′-fluoro uridine, and wherein the one or more thymidines are at the 3′ end of the first oligonucleotide.