Download RNA-Quant™ cDNA Synthesis Kit

Accelerating discoveries through innovations
Gene Analysis
RNA-Quant™ cDNA Synthesis Kit
Profile any RNA by real-time qPCR
An all-in-one cDNA synthesis kit
Highlights
For the last few decades of the 20th century, the underlying dogma of molecular
• Tail and tag all RNAs for cDNA
synthesis
biology has been that the purpose of RNA is to direct the assembly of proteins from
amino acids. These are the functions of messenger RNAs (mRNAs). mRNAs code for
• Obtain full RNA coverage for any
purpose
protein genes, have 5’ mG caps and most often have poly-A tails. A few exceptions to
this paradigm were known (for example, ribosomal RNA and transfer RNA, which are
• Profile any RNA
- mRNAs
- microRNAs
- sn/snoRNAs
- LncRNAs
- rRNAs
functional RNA macromolecules that do not code for protein, or viral genomes that
exist as or pass through an RNA phase as part of total genome replication). There are
now numerous exceptions that include: microRNAs, small nuclear RNAs (snRNAs),
small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs). The functions
of each class of RNA are briefly summarized in the Table below.
RNA-Quant allows you to profile all RNAs
Type of RNA
Functions
Messenger RNAs (mRNAs)
MicroRNAs (miRNAs)
Code for Proteins
Regulate mRNA translation
Bridge splicing events during
transcription
Guide methylation on target
RNAs
Bind chromatin for epigenome
changes and scaffolds for protein
complexes
Structural and catalytic subunits
of ribosomes
Small Nuclear RNAs (snRNAs)
Small Nucleolar RNAs
(snoRNAs)
Long Non-coding RNAs
(lncRNAs)
Ribosomal RNAs (rRNAs)
How the RNA-Quant Kit Works
10 pg–10 μg
total RNA
mRNAs
microRNAs
sn/snoRNAs
lncRNAs
rRNAs
polyA
polymerase
polyA-tailed 5’
lncRNAs
AAAAAAAAA 3’
Incubate at 37°C, 30 min
AAAAAAAAA 3’
NV T T T T T T T T T
5’
5’
Anneal adaptor
60°C, 5 min
5’
Generate comprehensive cDNA coverage
anchor-tailed
3’
Tag All RNAs
Anneal Adaptor
Any RNA
Convert to cDNA
3’
AAAAAAAAA 3’
TTTTTTTTT
5’
RT + random primers to make cDNA
42°C, 60 min
The poly-A tailing and tagging method in the RNA-Quant
cDNA pool of anchor-tailed RNAs
system generates cDNA that is highly unique and when used
3’
TTTTTTTTT
5’
with the 3’ Universal reverse primer does not produce any
signals in minus RT controls and does not detect any residual
genomic DNA. There is ZERO background in the minus RT
cDNA ready for
qPCR analysis
controls and the qPCR assays do not show any amplification
signals even with spiked-in genomic DNA. Profile with
confidence and only detect your RNAs of interest.
www.systembio.com/rna-quant
RNA-Quant™ cDNA Synthesis Kit
Efficiently make cDNA to measure any RNA by qPCR
Total RNA was harvested from human HT1080 cells using standard Trizol extraction protocols. The RNA-Quant kit was used
to tail and tag all RNAs into quantifiable cDNA for qPCR analysis. Sample amplification plots and specificity tests using
dissociation analyses are shown below.
GAPDH
Tm = 79˚C
U6
18S rRNA
mRNAs
sn/snoRNAs
rRNAs
miR-21
Tm = 71˚C
miR-18a
miR-21
MicroRNAs
RNU43
miR-18a
Tm = 71˚C
Lamin A/C
18S rRNA
Tm = 77˚C
GAPDH
miR-200c
miR-200c
Tm = 72˚C
U6 snRNA
Tm = 73˚C
Lamin A/C
Tm = 70˚C
RNU43
snoRNA
Tm = 75˚C
BC200
lncRNA
Tm = 80˚C
7SK
BC200
LncRNAs
7SK
lncRNA
Tm = 81˚C
Kit components - everything you need to start
40 l
5X PolyA Polymerase Buffer
10 l Poly A Reaction
10 l
PolyA Polymerase
(enough for 20 reactions)
20 l
25 mM MnCl2
30 l
5 mM ATP
10 l
Oligo dT Adaptor
20 l RT Reaction
80 l
5X Reverse Transcriptase Buffer + Random
Primer Mix
(enough for 20 reactions)
20 l
Reverse Transcriptase
30 l
0.1 M Dithiothreitol (DTT)
50 l
dNTP Mix
1.2 ml
RNase-free Water
600 l
3’ Universal Reverse primer
HOTAIR
HOTAIR
lncRNA
Tm = 83.6˚C
50 l
miR-16 microRNA qPCR assay, 10
M (human and
mouse)
50 l
Y RNA lncRNA qPCR assay, 10
M (human and
mouse)
50 l
GAPDH mRNA qPCR assay, 10
M (human and
mouse)
Kit comes
complete with
qPCR assays for:
• miR-16 microRNA
• Y RNA lncRNA
• GAPDH mRNA
Gain a more comprehensive view into
the transcriptome changes in your
model system with RNA-Quant.
We Also Offer Custom Services
System Biosciences offers a wide-range of custom services to support your research, allowing you to spend less time making tools, and more
time making discoveries. To learn more, visit our website at www.systembio.com/service or call us at 888-266-5066.
System Biosciences, Inc.
265 North Whisman Rd.
Mountain View, CA 94043
Toll Free: 888.266.5066
Fax: 650-968-2277
Email: [email protected]
www.systembio.com
© 2012 System Biosciences, Inc. All rights
reserved. System Biosciences and the System
Biosciences logo are trademarks of System
Biosciences, Inc.