How do you synthesize your dsRNA?

The novel dsRNA purification and library construction method, named FLDS, consists of cellulose column chromatography, the physical fragmentation of dsRNA, cDNA synthesis using a loop primer, and the PCR amplification of cDNA (). The purification of dsRNA was achieved by the repeated affinity purification of dsRNA using cellulose powder and the enzymatic removal of ssRNA and DNA. Purified dsRNAs were fragmented using ultrasound to retrieve all types of dsRNA viruses in order to apply the previously reported full-length dsRNA cloning method using a loop primer (). The full-length dsRNA cloning method requires overlapped cDNAs synthesized from both terminal ends for further cDNA amplification, and was only applicable to short dsRNA molecules. Reverse transcription was initiated from the ligated loop primer on both ends of the dsRNA fragment. cDNA was then thermally denatured to allow annealing of single-stranded cDNA with the complementary sequence in the 3′ terminal region. The single-stranded regions of annealed cDNA were filled in with DNA polymerase. The double-stranded cDNA derived from dsRNA was amplified by PCR with a single primer (PC2) in order to obtain sufficient cDNA to construct a sequencing library.

Thiscomplex initiates the RNA synthesis.

The entire DNA sequence necessary for thesynthesis of a functional polypeptide or RNA molecule.

PCR template method for dsRNA Synthesis

Total RNA was isolated from a diatom colony using the TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacturer’s protocol. The RNA fraction was treated with DNase I (Takara, Otsu, Japan). Double-stranded cDNA was synthesized from 2 μg of total RNA with random primers (9-mers) using a PrimeScript Double Strand cDNA Synthesis Kit (Takara). The resultant cDNA was quantified using a Qubit dsDNA HS Kit.

The dsRNA can be made from cDNA or genomic DNA ..

DsRNA was purified as described by Okada et al. with a few modifications (, ). Briefly, the microbial sample was disrupted in liquid nitrogen in a mortar and total nucleic acids were manually extracted. DsRNA was purified twice through a micro-spin column (empty Bio-spin column; Bio-Rad Laboratories, Inc., Hercules, CA, USA) containing cellulose powder (Cellulose D; ADVANTEC, Tokyo, Japan) to obtain pure dsRNA. The dsRNA eluted from cellulose powder in MQ water was treated with DNaseI (amplification grade, Invitrogen, Carlsbad, CA, USA) and S1 nuclease (Invitrogen) in nuclease buffer (57 mM CH3COONa, 9.5 mM MgCl2, 1.9 mM ZnSO4, and 189 mM NaCl) and was then incubated at 37°C for 2 h. The final concentrations of CH3COONa, MgCl2, ZnSO4, and NaCl were adjusted to 90 mM, 15 mM, 3 mM, and 300 mM, respectively. DsRNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA). A one-tenth volume of 10 × ShortCut buffer and 10 × MnCl provided with ShortCut RNase III (NEB Japan, Tokyo, Japan) was added to the dsRNA solution and fragmented by ultrasound at 4°C in Snap-Cap microTUBEs using a Covaris S220 (Woburn, MA, USA). The fragmentation conditions were as follows; run time 35 s, peak power 140.0 W, duty factor 2.0%, and 200 cycles/burst. Fragmented dsRNA was divided into two equal volumes, and maintained at 37°C with or without ShortCut RNase III (NEB). DsRNAs were then purified using a ZymoClean Gel RNA Recovery Kit (ZymoResearch, Orange, CA). Note that dsRNA purification from M. oryzae was carried out in the laboratory of Prof. Teraoka.

RNA polymerase I is dedicated to the synthesis of only one type of RNA molecule(pre-rRNA).
The 5' cap present on all eukaryoticmRNAs seems to be the first signal to start protein synthesis.

DRSC: Protocol for dsRNA Synthesis - diyhpl

The ORF A or ORF B domain of CHV1-EP721 was interchanged with those of CHV1-EP713 or CHV1-Euro7 with the aid of corresponding infectious cDNA clones of CHV1-EP713 () and CHV1-Euro7 () as described previously by Chen and Nuss (). The first group of chimeric viruses was configured with complete ORF A or ORF B domains swapped among CHV1-EP721, CHV1-Euro7, and CHV1-EP713. Briefly, between CHV1-EP721 and CHV1-EP713, the chimeric ORF A and ORF B junctions were first constructed by PCR so that the sequences of the domains representing target viruses were precisely configured. For chimera 721A713B, the junction contained the restriction sites SspI (position 2357) and SacI (position 3395), and for 713A721B, the junction contained the restriction sites HindIII (position 2284) and XhoI (position 3581). These restriction sites allowed the joining of the rest of the sequence of the 5′- and 3′-end portions of the target virus. Since the genome sequence from the SspI site in position 2357 to the end of ORF A is exactly the same between CHV1-EP721 and CHV1-Euro7, this restriction site was used to swap ORF A (NotI/SspI fragment) or ORF B (SspI/SpeI fragment) for making chimeric viruses. The second group of chimeric viruses was made by swapping specific domains within ORF B between CHV1-EP721 and CHV1-Euro7 by relying on common NarI (position 5311) and MluI (position 7843) restriction sites as illustrated in Fig. . The integrity of each chimera was examined by restriction pattern and sequence analysis of the junction of interchanged domains. The chimeric viral dsRNA from the corresponding fungal transfectants was further confirmed by reverse transcription-PCR (RT-PCR) and restriction analysis.

The 5’ UTRs of most mRNAs contain a consensus sequence of5’-CCAGCCAUG-3’ involved in the initiation of protein synthesis.

For a word document of these dsRNA synthesis ..

This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies.

A method is described for the construction of full-length cDNA clones of dsRNAs

Templates for dsRNA synthesis are generated by ..

Templates can be generated by PCR on cDNA (including ESTs from BDGP), genomic DNA, or first strand RT-cDNA. Most of the dsRNA should correspond to exons but dsRNA with two or more exons interrupted by introns will also work well. We generally aim for ~500 bp products although RNAi with products ranging from 150-3000bp have been shown to work. The target sequence should not contain complete 21-mer homology to other genes or your dsRNA could be non-specific. One case has been seen where the template contained one mismatch in a 21-mer identical stretch to another gene, and still only the level of the intended target was reduced, while the close homologue was unaffected. Since the targeted region within a transcript may influence the success of RNAi, the safest approach is to use dsRNA corresponding to the 5' or 3' UTR. It is recommended to check the various sequence sources (Publications, NCBI and BDGP, genomic and ESTs) to confirm the primary sequence of a given gene.