Methods and kits for sense RNA synthesis
Methods and kits are provided for performing multiple rounds of sense RNA synthesis. 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 performing multiple rounds of synthesis of at least one sRNA molecule, comprising:
a) providing at least one first round single stranded cDNA molecule having a 5′ end and a 3′ end;
b) attaching an oligodeoxynucleotide tail onto the 3′ end of said first round cDNA molecule;
c) annealing to said oligodeoxynucleotide tail a single stranded promoter template comprising a first RNA polymerase recognition sequence and at least a second different RNA polymerase recognition sequence 3′ to said first recognition sequence, wherein said single stranded promoter template is not extendable with DNA polymerase;
d) extending said oligodeoxynucleotide tail such that said single stranded promoter template is converted into a first RNA polymerase promoter and at least a second RNA polymerase promoter 3′ to said first promoter;
e) initiating a first round of RNA transcription using an RNA polymerase which recognizes said first RNA polymerase promoter to produce at least one first round sRNA molecule;
f) synthesizing at least one second round single stranded cDNA molecule from said first round sRNA molecule, said second round single stranded cDNA molecule having 5′ and 3′ ends;
g) degrading said first round sRNA molecule;
h) annealing a single stranded promoter oligonucleotide complementary to said second different RNA polymerase recognition sequence such that a second RNA polymerase promoter is formed; and
i) initiating a second round of RNA transcription using an RNA polymerase which recognizes said second RNA polymerase promoter to produce at least one second round sRNA molecule,
thereby performing multiple rounds of synthesis of at least one sRNA molecule and producing multiple sRNA copies.
2. The method of claim 1 , wherein a) comprises providing a RNA molecule having 5′ and 3′ ends; and synthesizing a single stranded cDNA molecule from said RNA molecule.
3. The method of claim 2 , wherein synthesis of the single stranded cDNA molecule comprises contacting the RNA molecule with a primer in the presence of a reverse transcriptase.
4. The method of claim 3 , wherein the primer is selected from the group consisting of oligodT primer, random primer, and combinations thereof.
5. The method of claim 4 , wherein the primer comprises a 5′ extension containing a specific nucleotide sequence.
6. The method of claim 4 , wherein the 3′ terminal nucleotide of the primer is not a substrate for terminal deoxynucleotide transferase but can be extended by reverse transcriptase.
7. The method of claim 6 , wherein the 3′ terminal nucleotide of the primer is a ribonucleotide.
8. The method of claim 4 , wherein the primer comprises a 5′ extension containing a specific nucleotide sequence, wherein the 3′ terminal nucleotide of said primer is a ribonucleotide.
9. The method of claim 1 , wherein the single stranded promoter template comprises a first RNA polymerase recognition sequence selected from the group consisting of T7, T3 and SP6 RNA polymerase recognition sequence, a second RNA polymerase recognition sequence selected from the group consisting of T7, T3 and SP6 RNA polymerase recognition sequence, and optionally a third RNA polymerase recognition sequence selected from the group consisting of T7, T3 and SP6 RNA polymerase recognition sequence, wherein said first, second and optionally third RNA polymerase recognition sequences are different.
10. The method of claim 1 , wherein f) comprises contacting the first round sRNA molecule with a primer in the presence of reverse transcriptase.
11. The method of claim 10 , wherein the reverse transcriptase is RNase H − .
12. The method of claim 10 , wherein the reverse transcriptase is RNase H + and wherein the RNase H + activity degrades the first round sRNA molecule in g).
13. The method of claim 10 , wherein reverse transcriptase activity in f) is inactivated prior to initiation of second round RNA transcription in i).
14. The method of claim 10 , wherein the primer is selected from the group consisting of oligodT primer, random primer, and combinations thereof.
15. The method of claim 5 , wherein f) comprises contacting the sRNA molecule with a second primer in the presence of reverse transcriptase, wherein said second primer comprises a nucleotide sequence corresponding to the specific nucleotide sequence contained in the 5′ extension of the first primer.
16. The method of claim 5 , wherein the primer is selected from the group consisting of oligodT primer, random primer, and combinations thereof.
17. The method of claim 1 , further comprising reverse transcribing the resulting multiple sRNA copies, thereby producing multiple single stranded cDNA molecules.
18. The method of claim 1 , further comprising adding polyA tails to the resulting multiple sRNA copies.
19. A method for performing multiple rounds of synthesis of at least one sRNA molecule, comprising:
a) providing at least one first round single stranded cDNA molecule having a 5′ end and a 3′ end;
b) attaching an oligodeoxynucleotide tail onto the 3′ end of said first round single stranded cDNA molecule;
c) annealing to said oligodeoxynucleotide tail a single stranded promoter template comprising a first RNA polymerase recognition sequence and at least a second different RNA polymerase recognition sequence 3′ to said first recognition sequence, wherein said single stranded promoter template is not extendable with DNA polymerase;
d) extending said oligodeoxynucleotide tail such that said single stranded promoter template is converted into a first RNA polymerase promoter and at least a second RNA polymerase promoter 3′ to said first promoter;
e) initiating a first round of RNA transcription using an RNA polymerase which recognizes said first RNA polymerase promoter to produce at least one first round sRNA molecule;
f) synthesizing at least one second round single stranded cDNA molecule from said first round sRNA molecule, said second round single stranded cDNA molecule having 5′ and 3′ ends;
g) degrading said first round sRNA molecule;
h) annealing excess single stranded promoter template from step c) to the 3′ end of said second round cDNA molecule;
i) extending the 3′ end of said second round cDNA molecule such that said excess single stranded promoter template is converted into a first RNA polymerase promoter and at least a second RNA polymerase promoter 3′ to said first promoter; and
j) initiating a second round of RNA transcription using an RNA polymerase which recognizes said first or second RNA polymerase promoter to produce at least one second round sRNA molecule,
thereby performing multiple rounds of synthesis of at least one sRNA molecule and producing multiple sRNA copies.
20. The method of claim 19 , wherein enzyme activity in i) is inactivated prior to initiation of second round RNA transcription in j).