morgan@go2biotech.comKR202-lnRcute lncRNA First-Strand cDNA Kit-171207
lnRcute lncRNA First-StrandcDNA Kit
Ultra-sensitive lncRNA specific reverse transcription reagent
lnRcute lncRNA First-Strand cDNA Kit
Cat. No. 4992785/4992908
Storage Conditions
Store at -30~-15°C for up to one year.
Product Description
lnRcute lncRNA First-Strand cDNA Kit is specially developed for reverse
transcription of long non-coding RNA (lncRNA). Compared with mRNA,
lncRNA has the characteristics of lower abundance, larger difference
in GC content, more complex secondary structure, etc., therefore it is
difficult to achieve the desired reverse transcription results of lncRNA
using traditional reverse transcription reagents. The lnRcute lncRNA FirstStrand cDNA Kit contains gDNase which can efficiently remove the residual
genomic DNA in RNA samples by incubating the RNA samples at 42°C for
3 minutes, and thus effectively avoid the interference of residual genomic
DNA on the subsequent PCR detection results. The reverse transcriptase
used in the lnR RT Enzyme Mix is FastKing RT Enzyme, which is a novel
reverse transcriptase with a hydrophobic motif added by molecular
modification, resulting in stronger RNA affinity and thermal stability, and
further improves its reverse transcription efficiency and reaction rate.
The synthesis of the first-strand cDNA can be completed in 15 minutes at
42°C, with at least 10 kb in length. In addition, the affinity between the
novel enzyme and RNA is stronger, and in combination with the specially
optimized buffer system and primer system, this kit is outstanding in the
aspects of stress resistance and effective reverse transcription of RNA
templates with high GC content, complex secondary structure and low
abundance. This kit is especially suitable for the reverse transcription
reaction of lncRNA with relatively low expression level and relatively
complex secondary structure.
Product Highlights
Excellent performance: The reverse transcription efficiency can reach
over 95%; The detection limit of total RNA can be as low as 10 ng, and the
reverse transcription length can reach 10 kb.
Simple and fast: The reaction system can be simply set up, and the lncRNA
first-strand cDNA can be synthesized within 21 minutes.
Wide applications: This kit can be widely used for RNA templates from
various species, and for RNA templates with large GC content difference,
complex secondary structure, low abundance and high level of impurities.
Good compatibility: Compatible with downstream applications such as RTqPCR with high sensitivity and good stability.
Precautions
- The following protocol is applicable to the total RNA with the template
amount of 10 ng-2 μg. If the total RNA amount is higher than 2 μg,
please increase the reaction system volume in proportion. - Please operate on ice to prevent RNA degradation.
- For RNA template with complex secondary structure, a denaturing step
is recommended, that is, before starting, incubate the template RNA at
65°C for 5 minutes, then transfer to ice quickly to perform the next step. - According to different experimental needs, the Oligo-dT Primer or Gene
Specific Primer can also be applied. The amount of primers is as follows:
Oligo-dT Primer 50 pmol/ 20 μl reaction system, Gene Specific Primer 5
pmol/ 20 μl reaction system. - The non-specific amplification effect during the PCR reaction can be
decreased by improving the reverse transcription reaction temperature
to 50°C.
Protocol
Rapid synthesis of lncRNA first-strand cDNA by lnRcute lncRNA FirstStrand cDNA Kit.
For 10 ng-2 μg total RNA, please set up a 20 μl reaction system. The
procedure is as follows:
- Thaw the template RNA on ice; Thaw 5×gDNA Buffer, lnR-RT Primer Mix,
10×lnR RT Buffer and RNase-Free ddH2
O at room temperature (15-25°C)
and quickly place on ice. Vortex the solutions before use, and pulse-spin
the tubes to collect the liquid on the tube wall.
Note: Please perform the following steps on ice. To ensure the
accuracy of the reaction mixture, it is recommended to prepare a
mastermix first, then aliquot into each reaction tube. - Prepare the genomic DNA removal reaction mix according to table 1, and
mix thoroughly by pipetting and pulse-spin the tube. Incubate at 42°C for
3 min, then quickly put on ice.
- Prepare the reverse transcription reaction mix according to table 2.
Table 2. Reverse transcription reaction system
- Transfer the reverse transcription reaction mix to the gDNA removal
reaction mix from step 2, and thoroughly mix to make a 20 μl
reaction system. - Incubate the reaction at 42°C for 15 min.
- Incubate the reaction at 95°C for 3 min, then quickly transfer to
ice. The cDNA product can be used for subsequent experiments, or
stored at -20°C.
RNA templates requirements
Reverse transcriptase uses RNA as template to synthesize the first strand
of cDNA. The quality and input amount of template RNA directly affect the
results of reverse transcription.
- Integrity of the template: The integrity of the template RNA is very
important for reverse transcription. The remaining RNase in the RNA
template will degrade the template RNA, resulting in low yield of cDNA
products or even no cDNA products. - Purity of the template: Protein, salt ion, EDTA, ethanol, phenol or other
impurities remaining in the RNA template will affect the activity of
reverse transcriptase, thus affect the results of reverse transcription. - Input amount of the template: For 10 ng-2 μg of input template RNA,
the above protocol is applicable. If the amount of template RNA is more
than 2 μg, please increase the reaction system volume in proportion.
Important notes
- If the follow-up experiment is RT-qPCR, the input amount of RT products
should not exceed 1/10 of the final volume of PCR reaction system.
For example, for 50 μl PCR reaction system, the input amount of RT
products should not exceed 5 μl. - Upon the completion of reverse transcription, please place the cDNA
products on ice before preparing the subsequent PCR reaction system.
For long-term storage, please store at – 20°C.
KR202-lnRcute lncRNA First-Strand cDNA Kit-171207
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