Method for producing a sense RNA molecule
Methods and kits are provided for producing sense RNA molecules. The sense RNA molecules can be used in various research and diagnostic applications, such as gene expression studies involving nucleic acid microarrays.
1. A method for producing a sense RNA molecule, comprising:
a) providing a single stranded cDNA molecule having 5′ and 3′ ends;
b) attaching a homopolymeric tail for annealing a single stranded promoter template to the 3′ end of said single stranded cDNA molecule using terminal deoxynucleotidyl transferase;
c) annealing a single stranded promoter template consisting essentially of a single RNA polymerase promoter sequence at a 5′ end and a sequence complementary to the homopolymeric tail at a 3′ end thereof to said homopolymeric tail through base pairing with the complementary sequence of the single stranded promoter template, wherein said single stranded promoter template is not extendable with DNA polymerase;
d) extending said homopolymeric tail such that said single stranded promoter template is converted to a double stranded promoter; and
e) initiating RNA transcription using an RNA polymerase which recognizes said double stranded promoter,
thereby producing a sense RNA (sRNA) molecule.
2. The method of claim 1 , wherein a) comprises providing a mRNA transcript having 5′ and 3′ ends; and synthesizing a single stranded cDNA molecule from said mRNA transcript.
3. The method of claim 2 , wherein synthesis of the single stranded cDNA molecule comprises reacting the mRNA molecule with a RNase H − reverse trancriptase.
4. The method of claim 3 , wherein synthesis of the single stranded cDNA molecule further comprises reacting the mRNA molecule with an oligodT primer.
5. The method of claim 3 , wherein synthesis of the single stranded cDNA molecule further comprises reacting the mRNA molecule with a random-primer.
6. The method of claim 2 , further comprising purifying the single stranded cDNA molecule prior to attaching the homopolymeric tail.
7. The method of claim 6 , further comprising degrading the mRNA transcript prior to purifying the single stranded cDNA molecule.
8. The method of claim 2 , wherein the mRNA-transcript is of mammalian origin.
9. The method of claim 2 , wherein the mRNA transcript is of human origin.
10. The method of claim 2 , wherein the mRNA transcript is isolated from a biological source comprising degraded RNA.
11. The method of claim 1 , wherein the homopolymeric tail is a polydT tail.
12. The method of claim 1 , wherein the single stranded promoter template comprises a T7, T3 or SP6 RNA Polymerase promoter.
13. The method of claim 1 , wherein the single stranded promoter template is blocked at the 3′ end by a 3′ amino modifier, a 3′ deoxyterminator, or a 3′ dideoxyterminator.
14. The method of claim 1 , further comprising synthesizing second strand cDNA using a random primer, thus producing random-primed second strand cDNA fragments prior to initiating RNA transcription.
15. The method of claim 14 , wherein the random-primed second strand cDNA fragments are ligated together prior to initiating RNA transcription.
16. The method of claim 1 , wherein RNA transcription is initiated using a T7, T3 or SP6 RNA polymerase.
17. The method of claim 1 , further comprising amplifying the resulting sRNA molecule.
18. The method of claim 17 , wherein sRNA amplification is initiated using a combination of oligodT and random primers.
19. The method of claim 17 , wherein sRNA amplification is initiated using oligodT primers, and the resulting sRNA molecule comprises a polyA tail.
20. The method of claim 17 , wherein the sRNA amplification is initiated using random primers.
21. The method of claim 1 , further comprising adding a polyA tail to the resulting sRNA molecule.
22. The method of claim 21 , wherein the polyA tail is added using polyA polymerase.
23. The method of claim 1 , further comprising reverse transcribing the resulting sRNA molecule, thereby producing a single stranded cDNA molecule.
24. The method of claim 23 , wherein the reverse transcription comprises incorporating detectably labeled nucleotides into the single stranded cDNA molecule.
25. The method of claim 24 , wherein the detectably labeled nucleotides comprise a fluorescent dye.
26. The method of claim 25 , wherein the fluorescent dye is cy3 or cy5.
27. The method of claim 23 , further comprising attaching at least one detectable label to the resulting cDNA molecule.
28. The method of claim 27 , wherein the detectable label is attached to the cDNA molecule via a nucleic acid dendrimer.
29. The method of claim 1 , wherein a) comprises providing a mRNA molecule having 5′ and 3′ ends; and synthesizing a single stranded cDNA molecule from said mRNA molecule.
30. The method of claim 29 , wherein a) further comprises adding a polyA tail to the cDNA molecule.
31. The method of claim 30 , wherein the polyA tail is added using polyA polymerase.
32. The method of claim 31 , wherein synthesis of the single stranded cDNA molecule further comprises reacting the mRNA molecule with an oligodT primer.