Biomedical Engineering Reference
In-Depth Information
Table 1
List of Primers and Probes
for Real-Time Quantitative Polymerase Chain Reaction
Forward/
reverse/
Amplicon
Name
Sequence
probe
size (bp)
EBER82F
5'-GAGAGGCTTCCCGCCTAGA-3'
Forward
82
EBER181F
5'-TACATCAAACAGGACAGCCGTT-3'
Forward
181
EBER294F
5'-TCCCGCAGTTCCACCTAAAC-3'
Forward
294
EBER385F
5'-GACTCTGCTTTCTGCCGTCTTC-3'
Forward
385
EBER493F
5'-CTACGCTGCCCTAGAGGTTTTG-3'
Forward
493
EBER584F
5'-ACACACCAACTATAGCAAACCCC-3'
Forward
584
EBER695F
5'-TCCCAGAGAGGGTAAAAGAGGG-3'
Forward
695
EBER781F
5'-CCCGCTACGTGCAGTGCT-3'
Forward
781
EBER891F
5'-TACAGCTAAATGCCCACCA-3'
Forward
891
EBER1000F
5'-GCAGAGGACATTGGGCAGGT-3'
Forward
1000
EBER-R 5'-AAATAGCGGACAAGCCGAATA-3' Reverse
EBERU3T 5'-(FAM)TCTCCCAGAGGGATTAGA(MGBNFQ)-3' Probe
EBER, Epstein-Barr virus-encoded RNAs; FAM, 6-carboxyfluorescein; MGBNFQ, minor
groove-binding nonfluorogenic quencher.
3.2. DNA Extraction
For the quantitative analysis of plasma DNA, the same plasma sample would
be subjected to multiple real-time quantitative PCRs of different amplicon
sizes, and the number of analysis depends on the size range and the resolution
of the size distribution. Therefore, a higher elution volume is necessary in or-
der to give adequate material for the whole panel of analyses. Thus, we have
been using the QIAamp DNA Midi Kit (Qiagen, Hilden, Germany) for DNA
extraction from plasma for the size analysis of circulating DNA.
1.
Follow the “blood and body fluid protocol” as per manufacturer's instructions.
2.
2 mL plasma is loaded to the column and eluted with 300
µ
L dH 2 O ( 11 ) .
3.3. Real-Time PCR Assays
3.3.1. Design of TaqMan Assays
1.
The 10 PCR assays shared a common forward primer and a common TaqMan
probe, but consisted of 10 different reverse primers ( 11 ) .
2.
The locations of the reverse primers are determined by the desired amplicon
lengths.
3.
Although the forward primer and the probe are used by all reactions, it would be
more convenient if the melting temperatures of all the primers are approximately
 
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