System for providing control reactions for real time RT-PCR
Provided are methods and oligonucleotides useful as primers and templates for internal controls designed for use in Real Time Reverse Transcriptase Polymerase Chain Reactions. Use of the present methods and oligonucleotides allows validation of assay parameters and of the results that an assay set.
1. A multi-well PCR array, comprising:
(a) in a first well, a primer pair designed for amplifying a Genomic DNA Contamination Control (GDC) sequence, wherein
when the GDC sequence is a human genomic DNA sequence, one primer of the primer pair comprises SEQ ID NO:7 and the other primer of the primer pair comprises SEQ ID NO:8, or one primer of the primer pair comprises SEQ ID NO:9 and the other primer of the primer pair comprises SEQ ID NO:10;
when the GDC sequence is a mouse genomic DNA sequence, one primer of the primer pair comprises SEQ ID NO:13 and the other primer of the primer pair comprises SEQ ID NO:14; or
when the GDC sequence is rat genomic DNA sequence, one primer of the primer pair comprises SEQ ID NO:15 and the other primer comprises SEQ ID NO:16; and
(b) in a second well, a Plate Positive Control (PPC) template nucleic acid and a primer pair designed for amplifying the PPC template, wherein one primer of the primer pair designed for amplifying the PPC template nucleic acid comprises SEQ ID NO: 4 and the other primer of the primer pair designed for amplifying the PPC template nucleic acid comprises SEQ ID NO:5.
2. The multi-well PCR array of claim 1 , wherein the PPC template nucleic acid comprises SEQ ID NO:6.
3. The multi-well PCR array of claim 1 , further comprising:
(c) in a third well, a primer pair designed for amplifying the cDNA of a Reverse Transcription Control (RTC) RNA.
4. The multi-well PCR array of claim 3 , wherein one primer of the primer pair designed for amplifying the cDNA of the RTC RNA comprises SEQ ID NO:1 and the other primer of the primer pair designed for amplifying the cDNA of the RTC RNA comprises SEQ ID NO:2.
5. The multi-well PCR array of claim 3 , further comprising:
(d) in the fourth well, a primer pair designed for amplifying a house keeping gene (HKG).
6. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a human genomic DNA sequence, the HKG is selected from the group consisting of human beta-2-microglobulin (B2M), hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ribosomal protein L 13a (RPL 13A), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and actin, beta (ACTB) genes.
7. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a human genomic DNA sequence, the primer pair designed for amplifying the HKG comprises SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, or SEQ ID NO:25 and SEQ ID NO:26.
8. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a mouse genomic DNA sequence, the HKG is selected from the group consisting of mouse glucuronidase, beta (Gusb), hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), heat shock protein 90 kpa alpha class B member 1 (Hsp90ab1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and actin, beta (ACTB) genes.
9. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a mouse genomic DNA sequence, the primer pair designed for amplifying the HKG comprises SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, or SEQ ID NO:35 and SEQ ID NO:36.
10. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a rat genomic DNA sequence, the HKG is selected from the group consisting of rat ribosomal protein, large P1 (Rplp1), hypoxanthine guanine phosphoribosyl transferase (HPRT), ribosomal protein L 13a (Rpl13A), lactate dehydrogenase A (Ldha), and actin, beta (Actb) genes.
11. The multi-well PCR array of claim 5 , wherein when the GDC sequence is a rat genomic DNA sequence, the primer pair designed for amplifying the HKG comprises SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, or SEQ ID NO:45 and SEQ ID NO:46.