Biomedical Engineering Reference
In-Depth Information
unity indicates high integrity, whereas anything greater than five suggests degradation. The
assay is designed as a triplex assay using a labeled hydrolysis probe for detection of each
assay such that each amplicon is detected by a target-specific, differentially labeled probe.
PROTOCOL 6.1 Analysis of mRNA Integrity
Equipment and reagents
•
Reverse transcription
—reverse transcriptase enzyme (RT) 50 U/
μ
l
—10
×
RT buffer (supplied with RT)
—oligo-dT primer (500 ng/
μ
l)
—100m
M
dNTP mix (25 mM each of dATP, dCTP, dGTP and dTTP)
•
qPCR
—six GAPDH primers, two each for 5
,centerand3
amplicons (10
μ
M
each)
—three GAPDH probes, one each for 5
,centerand3
amplicons (5
μ
M
each)
—2
×
commercial qPCR master mix buffer (containing dNTPs and thermostable
a
polymerase) without MgCl
2
—25m
M
MgCl
2
—yeast tRNA (Invitrogen)
—universal RNA (Stratagene human, mouse, rat)
—real-time thermocycler with the capacity to detect multiplex reactions (e.g. Corbett
6000 or Stratagene MX3005p).
Oligonucleotides (5
-3
)
5
-GAPDH:
P:
(FAM)-
CCTCAAGATCATCAGCAATGCCTCCTG-(
BHQ1
)
F: GTGAACCATGAGAAGTATGACAAC
R: CATGAGTCCTTCCACGATACC
Center GAPDH:
P:
(HEX)-
CCTGGTATGACAACGAATTTGGCTACAGC-(
BHQ1
)
F: TCAACGACCACTTTGTCAAGC
R: CCAGGGGTCTTACTCCTTGG
3
-GAPDH:
P:
(CY5)-
CCCACCACACTGAATCTCCCCTCCT-(
BHQ3
)
F: AGTCCCTGCCACACTCAG
R: TACTTTATTGATGGTACATGACAAGG
