qRT-PCR
Item
miR-16-5p TaqMan assay
let-7a-5p TaqMan assay
miR-223-3p TaqMan assay
TaqMan MicroRNA Reverse Transcription Kit
18S rRNA TaqMan assay, FAM
GAPDH TaqMan assay, FAM
BRAF TaqMan assay, FAM
Universal RNA Spike I, FAM
High Capacity cDNA Reverse Transcription Kit, with RNase Inhibitor
TaqMan 2X Universal PCR Master Mix, no AmpErase UNG
qPCR machine
96-well plates compatible with qPCR machine
Vendor
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
TATAA Biocenter
Life Technologies
Life Technologies
Catalog #
4427975
4427975
4427975
4366596
4331182
4331182
4331182
RS25PFI
4374966
4324018
Assay#
000391
000377
002295
target
miRNA
miRNA
miRNA
miRNA
Hs99999901_s1 mRNA
Hs02758991_g1 mRNA
Hs00269944_m1 mRNA
mRNA
mRNA
miRNA and mRNA
miRNA and mRNA
miRNA and mRNA
For miRNAs
1.1
Normalize input RNA samples to a concentration of 1 ng/ul.
1.2
Thaw RT kit components on ice. Mix gently and centrifuge briefly.
1.3
Calculate number of RT reactions, including no-input control.
1.4
Make RT master mix on ice, using attached spreadsheet to scale the following per
reaction mixture:
Component
100mM dNTPs (with dTTP)
MultiScribe™ Reverse Transcriptase, 50 U/μL
10✕ Reverse Transcription Buffer
RNase Inhibitor, 20 U/μL
Nuclease-free water
Total volume
1.5
1.6
Volume per 15-μL reaction
0.15 μL
1.00 μL
1.50 μL
0.19 μL
8.16 μL
11.00 μL
Mix and centrifuge briefly.
Combine in 0.2 ml qPCR well:
1 ul input RNA
11 ul RT master mix.
3 ul 5X RT primer
1.7
Seal, mix, and centrifuge briefly.
1.8
Incubate on ice for 5 minutes.
1.9
Place in thermocycler and run the following program:
16 oC 30 minutes
42 oC 30 minutes
85 oC 5 minutes
4 oC Hold
The reactions may be stored at -20 oC at this point.
1.10 Thaw frozen PCR kit components on ice. Mix and centrifuge briefly.
1.11 Swirl mastermix bottle gently to mix.
1.12 Calculate number of samples, including no-input and no-template controls for each
miRNA assay.
1.13 Set up triplicate PCR reactions for each template/assay combination, using the
attached spreadsheet to scale the following per reaction mixture:
Component
TaqMan® Small RNA Assay (20✕)
Product from RT reaction
TaqMan® Universal PCR Master Mix II (2✕), no UNG
Nuclease-free water
Total volume
1.14
1.15
1.16
1.17
Volume per 10-μL reaction
0.5 μL
0.66 μL
5 μL
3.84 μL
10.00 μL
Mix and centrifuge briefly.
Distribute into 0.2 ml qPCR wells, 10 ul per well.
Seal, mix, and centrifuge briefly.
Place in qPCR machine and run the following program:
1 cycle of: 95 oC 10 minutes
40 cycles of: 95 oC 15 seconds
60 oC 60 seconds
For mRNAs
2.1
Normalize input RNA samples to a concentration of 1 ng/ul.
2.2
Thaw RT kit components on ice. Mix gently and centrifuge briefly.
2.3
Calculate number of RT reactions, including no-input control.
2.4
Make RT master mix on ice, using attached spreadsheet to scale the following per
reaction mixture:
Component
Volume per 10-μL reaction
10✕ RT Buffer
1.0 μL
25✕ dNTP Mix (100 mM)
0.4 μL
10✕ RT Random Primers
1.0 μL
MultiScribe™ Reverse Transcriptase
0.5 μL
RNase Inhibitor
0.5 μL
Universal RNA Spike I template
1.0 μL
Nuclease-free H2O
4.6 μL
Total per Reaction
9.0 μL
2.5
Mix and centrifuge briefly.
2.6
Combine in 0.2 ml qPCR well:
1 ul input RNA
9 ul RT master mix.
2.7
Seal, mix, and centrifuge briefly.
2.8
Keep on ice until you are ready to put into thermocycler.
2.9
Place in thermocycler and run the following program:
25 oC 10 minutes
37 oC 120 minutes
85 oC 5 minutes
4 oC Hold
The reactions may be stored at -20 oC at this point.
2.10 Thaw frozen PCR kit components on ice. Mix and centrifuge briefly.
2.11 Swirl mastermix bottle gently to mix.
2.12 Calculate number of samples, including no-input and no-template controls for each
miRNA assay.
2.13 Set up triplicate PCR reactions for each template/assay combination, using the
attached spreadsheet to scale the following per reaction mixture:
*For the TaqMan Assays, set up this reaction mixture:
Component
20X TaqMan® Gene Expression Assay
Product from RT reaction
TaqMan® Universal PCR Master Mix II (2✕), no UNG
Nuclease-free water
Total volume
Volume per 10-μL reaction
0.5 μL
1 μL
5 μL
3.5 μL
10.00 μL
*For the Universal RNA Spike I Assay, set up this reaction mixture:
Component
Volume per 10-μL reaction
Spike I Assay primers
0.4 μL
Spike I Assay probe
0.2 μL
Product from RT reaction
1 μL
TaqMan® Universal PCR Master Mix II (2✕), no UNG
5 μL
Nuclease-free water
3.4 μL
Total volume
10.00 μL
2.14
2.15
2.16
2.17
Mix and centrifuge briefly.
Distribute into 0.2 ml qPCR wells, 10 ul per well.
Seal, mix, and centrifuge briefly.
Place in qPCR machine and run the following program:
1 cycle of: 50 oC 2 minutes
95 oC 10 minutes
40 cycles of: 95 oC 15 seconds
60 oC 60 seconds