G3PDH genes (500 bp) were amplified by PCR using cDNA templates that were synthesized with various RNase H minus RTases from G3PDH mRNA (102-105 copies/reaction). The RTase reaction was performed with specific reverse primers and 100 U enzyme at 42ºC for 20 min. The results indicated that ReverTra Ace® is suitable for RT-PCR amplifications that require sensitivity.
Normalized cDNA corresponding to genes expressed in flowers and inflorescences of F. esculentum and F. tataricum was sequenced using the 454 pyrosequencing technology. This resulted in 267 (for F. esculentum) and 229 (F. tataricum) thousands of reads with average length of 341-349 nucleotides. De novo assembly of the reads produced about 25 thousands of contigs for each species, with 7.5-8.2× coverage. Comparative analysis of two transcriptomes demonstrated their overall similarity but also revealed genes that are presumably differentially expressed. Among them are retrotransposon genes and genes involved in sugar biosynthesis and metabolism. Thirteen single-copy genes were used for phylogenetic analysis; the resulting trees are largely consistent with those inferred from multigenic plastid datasets. The sister relationships of the Caryophyllales and asterids now gained high support from nuclear gene sequences.
RNA/Reverse Transcription (RT) & cDNA Synthesis …
In our study we tried to minimize the influence of technical effects. DNAse treatment of RNA was performed before cDNA synthesis to preclude genomic DNA contamination; also low quality data were excluded from the assembly process. As for the assembly accuracy, in the absence of a reference genome the possibility of assembly errors can not be completely ruled out. However given the relatively large length of 454-generated reads even chimeric combination of reads corresponding to different genes in one contig is unlikely to hamper efficient BLAST search. Indeed, assembly errors cause problems with similarity search only if short reads corresponding to small and weakly conserved fragments of different genes are combined into one contig.
MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit; ..
In this webinar, Dr. Rachel Fish (Clontech Laboratories, Inc.) presents data generated with . This kit is a complete system designed to generate cDNA from enriched total RNA, allowing researchers to improve sequence coverage, identify rare gene fusions, and save costs with a targeted RNA-seq approach.
DirectScript cDNA Synthesis Kit ..
In this webinar, Dr. Magnolia Bostick (Clontech Laboratories, Inc.) presents data showing how the complements random-primed SMARTer cDNA synthesis from low-input RNA samples, including RNA isolated from FFPE tissues.
MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit;
In this webinar, Dr. Magnolia Bostick (Clontech Laboratories, Inc.) discusses the and the . These kits allow you to generate cDNA from low-input or degraded total RNA samples and sequence on either Illumina or Ion Torrent platforms.
Total RNA Isolation and cDNA synthesis from Bixa …
In this webinar, Dr. Andrew Farmer (Vice President of R&D, Clontech Laboritories, Inc.) explains how the SMARTer method of cDNA synthesis facilitates studies of transcriptome complexity from very small samples (1 to 1,000 cells, or 10 pg to 10 ng of total RNA).