RevertAid™ Premium
First Strand cDNA Synthesis Kit
#K1651, #K1652
CERTIFICATE OF ANALYSIS
#K1651
Lot ___
QUALITY CONTROL
制作南瓜灯RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generated a prominent
496 bp product on 1% agaro gel after ethidium bromide staining.
Quality authorized by: Jurgita Zilinskiene
Rev.3
IMPORTANT NOTES RNA quantity
Avoiding ribonuclea contamination ∙U 1 pg - 5 µg of total RNA, 0.1 pg - 500 ng of poly(A) mRNA or 0.01 pg – 500 ng of
specific RNA transcript to generate first strand cDNA as the initial step of a two-step RTRNA purity and integrity is esntial for synthesis of full-length cDNA. RNA can be degraded
PCR protocol.
by RNa A, which is a highly stable contaminant found in any laboratory environment. All
components of the kit have been rigorously tested to ensure that they are RNa free. To ∙U 1 µg of isolated mRNA to generate first strand cDNA for cond-strand synthesis and prevent contamination both the laboratory environment and all prepared solutions must be free subquent cloning reactions.premium是什么意思
of RNas.
Primers
General recommendations to avoid RNa contamination:
agrarian
Synthesis of first strand cDNA can be primed with either oligo(dT)18 primer, random primers or ∙U certified nuclea-free labware or DEPC-treat all tubes and pipette tips to be ud in
gene-specific primers.
tabercDNA synthesis.
Oligo(dT)18 primes cDNA synthesis from the poly(A) tail prent at the 3’-end of eukaryotic
∙Wear gloves when handling RNA and all reagents, as skin is a common source of RNas.
mRNA. Random primers initiate cDNA synthesis from the total RNA population (rRNA and Change gloves frequently.
mRNA). Therefore, using random primers for first strand synthesis results in a greater
∙U RNa-free reagents, including high quality water (e.g., Water, nuclea-free, complexity of the generated cDNA compared with the oligo(dT)
18 primer. As a conquence,
#R0581). the nsitivity and specificity of subquent PCR reactions may be reduced. However, there ∙Keep the kit components tightly aled when not in u. Keep all tubes tightly clod during are veral applications where it is beneficial to u random primers, such as cDNA synthesis the rever transcription reaction. using mRNAs without a poly(A) tail, or cDNA synthesis using poly(A)-enriched RNA samples.
Gene-specific primers are ud to synthesize specific cDNA from a pool of total RNA or mRNA Template RNA
and must be obtained by the ur.
东京大学世界排名Total cellular RNA isolated by standard methods is suitable for u with the kit. Purified RNA
must be free of salts, metal ions, ethanol and phenol to avoid inhibiting the cDNA synthesis
reaction. CONTROL REACTIONS for RT-PCR / RT-qPCR
For RT-PCR applications, template RNA must be free of DNA contamination. Prior to cDNA The following negative control reactions should be ud to verify the results of the first strand synthesis, RNA can be treated with DNa I, RNa-free (#EN0521) to remove trace amounts cDNA synthesis.
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of DNA. ∙Rever transcripta minus (RT-) negative control is important in RT-PCR and Always perform a RT-minus negative control reaction, which includes all components for RT-qPCR reactions to asss for genomic DNA contamination of the RNA sample. The RT-PCR except the RevertAid™ Premium Enzyme Mix. control RT- reaction should contain all reagents for the rever transcription reaction RNA sample quality except for the RevertAid™ Premium Enzyme Mix.
∙No template control (NTC) is important to asss for reagent contamination. The NTC Asss RNA integrity prior to cDNA synthesis. Total eukaryotic RNA can be analyzed by
reaction should contain all reagents necessary for the rever transcription reaction except agaro gel electrophoresis followed by ethidium bromide staining. The RNA isglobe是什么意思
for the RNA template.
considered to be intact, if both 18S and 28S rRNA appear as sharp bands after
electrophoresis of total RNA. The 28S rRNA band should be approximately twice as
inten as the 18S rRNA. Any smearing of rRNA bands is an indication of degraded
mRNA. If this occurs, a new sample of total RNA should be prepared. Alternatively, total
RNA can be analyzed using a bioanalyzer (e.g., Agilent 2100) which provides
quantitative information about the general state of the RNA sample, the RNA integrity
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吕布与貂蝉的故事number (2). A reference gene/target gene 3’:5’ integrity assay (3) can also be ud to
determine the integrity of the RNA sample.
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