Chum-RNA allows cDNA synthesis from a single-cell quantity ..
The ProtoScript First Strand cDNA Synthesis Kit is contains everything needed for cDNA synthesis
Determination of the minimum amount of mRNA required for …
We also assessed the reproducibility of the cDNA synthesis method using the NuGEN-Ovation® RNA-Seq System by calculating the pairwise Spearman's correlation coefficient (R) of transcript abundances between biological replicates (four mouse sample 0 Gy and four mouse samples 2Gy) using data obtained from SOLiD and Illumina. By comparing transcript levels across libraries, we found high reproducibility among the replicates (R=0.9535 for 0 Gy replicates and R=0.9634 for 2 Gy replicates) in SOLiD data (A and B). Similarly, Spearman's correlation was good between biological replicates in Illumina data (data not shown). Furthermore, the SOLiD data set showed higher correlations between combined data sets (R=0.9634 for 0 Gy combined data set of two biological replicates and R=0.9685 for 2 Gy combined data set of two biological replicates), as shown in C and D, which illustrates the benefits of collecting replicated RNA sequencing data as has been previously supported by simulation (). The correlation coefficient of replicates proved that this method performed equally as well as previously published protocols (). However, the correlation for replicates between the two platforms was lower than the correlation between biological replicates using the same platform; this may be the result of differences in the two sequencing technologies. Spearman's correlation heat map is shown in .
tea-test on Nov 28 2009, 12:09 AM said:
Thankyou very much for a prompt reply,
this solve major part of my query..... but something still bugging me is that, isnt mRNA just about 5 to 10 percent of total RNA, will it be still sufficient? and one more thing you had mentioned 5 to 20ng of cDNA..... i did not quantify the cDNA..i quantified the RNA and based on this i mentioned the ug quantities, so how can i be sure what is the concentration of cDNA?
but once again thank you very much!!!
of RNA into all two-step cDNA synthesis ..
I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract RNA from my samples, I proceed with cDNA synthesis right away without measuring the RNA concentration or without normalizing equal amount of RNA for all my samples. It is only after making cDNA that I measure the concentration and add around 100 ng in each PCR tube.
my results are a bit funny now and I get my first signal after 35 cycles and surprisingly the samples that should give signal sooner are always the last ones. my friends are suggesting to measure my RNA before proceeding with cDNA synthesis. They say maybe too much RNA in the sample does not let cDNA to be properly synthesized !?
is this comment true?
I would even say that this is far too much input material, even for convential PCR this is pretty much. Typical amounts for real time PCR are ranging from 5 to 20 ng of cDNA. Putting too much cDNA into the reaction is 1) a waste of material and 2) it increases the possibility of PCR inhibition by coisolated impurities or salts from the RT reaction. Especially with RNA from blood I would be very cautious as heme is a potent PCR inhibitor. I would recommend to do first a serial dilution curve with your cDNA starting with 100ng down to eg. 0.001ng, depends on how strong your target gene is expressed. Then you will see if you have any inhibition effect.
With input amounts >100ng I don't get any amplification in 10µl volumes and I'm also using the ABI high capacity kit.
protocol: RNA-seq Library Preparation
RNA sequencing approaches to transcriptome analysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptome analysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500pg) of total RNA. We compared a single-sample library prepared by this Ovation® RNA-Seq system with two available methods of mRNA enrichment (TruSeq™ poly-A enrichment and RiboMinus™ rRNA depletion). Using the Ovation® preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (
Many RTs are available from commercial suppliers. and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. lacks 3´ → 5´ exonuclease activity. is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
Biomiga, FS01-01 – 1st Strand cDNA Synthesis System, …
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of total RNA for cDNA synthesis
First-strand cDNA Synthesis.
cDNA synthesis from the isolated RNA is apparently limited ..
Determination of the minimum amount of mRNA required for chum-RNA …
Buy Biomiga FS01-01, 1st Strand cDNA Synthesis System, …
rRNA contamination, the major challenge during transcriptome sequencing, decreases the sequencing depth for mRNA and adds to costs for unusable rRNA reads. Both methods of mRNA enrichment (poly-A selection of mRNA and rRNA depletion) require large amounts of total RNA as input material that may not be available in certain research areas, such as stem cells and cancer. Using the Ovation® RNA-Seq System, the total RNA input from a single sample without any enrichment generated lower rRNA reads (A and Supplementary Table S1. Further, our analysis of 92.5 million mapped short read sequences of 50 bases for cDNA libraries using the SOLiD platform and 38.5 million mapped paired end reads (2×50 bases) from same set of eight mouse samples using Illumina Genome Analyzer IIx revealed substantial enrichment of reads for mRNA and negligible rRNA reads when prepared with the Ovation® RNA-Seq System (1.92 and 2.09% for SOLiD and Illumina platforms, respectively) A and B. Generally, using routine cDNA synthesis protocols in cases where there is no depletion of rRNA before cDNA synthesis, RNA-seq data maps to rRNA >75% of the time for libraries from both prokaryotes and eukaryotes (,). The rRNA reads were reduced to 13% by using selective hexamer primers (low binding to rRNA), but this approach is quite costly, requiring 749 hexamers (). In our study, the alignment of ). A small proportion of reads (~2%) has also been mapped to rRNA in poly (dT) based on direct RNA sequencing and this further suggests that a small fraction of rRNA are polyadenylated ().
first-strand cDNA synthesis efficiency
To examine platform-specific biases, we used the Ovation® RNA-Seq System to prepare cDNA libraries for eight mouse testis tissue samples and sequenced these on two different next-generation sequencing platforms for a side-by-side comparison. We obtained 169.5 million reads from SOLiD sequencing and 61.5 million reads from two lanes of Illumina for the eight-mouse cDNA libraries. From these, we mapped 92.5 million (54.57%) and 38.5 million (62.6%) reads, respectively, to mouse genome (A and B). Briefly, 20.27% of these reads mapped unambiguously to known mouse transcripts (refSeq transcripts, 27 582), 2.35% to exon–exon junctions, 7.76% to exon–intron junctions, 36% to intergenic regions (outside of any known annotation) and of the remaining reads mapped to introns in SOLiD data (). In Illumina data, 15.28% of these reads mapped unambiguously to known mouse transcripts (refSeq transcripts, 27 582), 2.26% to exon–exon junctions, 5.62% to exon–intron junctions, 42.01% to intergenic regions (outside of any known annotation) and the remaining reads mapped to introns (). The mapping percentages of reads to exonic regions were two and three times more in Illumina and SOLiD data respectively in comparison to data published previously (). However, we have more intergenic reads in the data of both platforms comparatively which may reflect incomplete annotation of the mouse genome in refSeq; this percent of intergenic reads is also evident in literature for other RNA-seq methods and high-density arrays, and cloning/sequencing techniques (,).
ProtoScript® First Strand cDNA Synthesis Kit | NEB
Reads were initially mapped to ribosomal RNA sequences (5, 5.8, 12, 16, 18 and 28s) using Bowtie () with default settings. Reads that mapped to ribosomal sequences were excluded from further analysis. In the case of paired-end Illumina reads, both pairs were removed if either pair mapped to rRNA. Ribosomal RNA sequences were acquired from GenBank (,). Remaining reads were mapped to the genome using TopHat v.1.1.3 (). For single-end SOLiD reads, all other parameters were kept to TopHat default values. For paired-end reads, the mean insert sizes as determined by bioanalyzer were employed in TopHat mapping. The standard deviation of insert length was set to 50bp for all samples. Only uniquely mapped reads were recorded and used for downstream analysis in both paired and unpaired data.
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