cDNA library preparation |
The ultimate goal of genome projects is to produce a complete accurate sequence of the entire genetic material of a biological species. The part of the genome expressed as mRNA, often referred to as the transcriptome, contains much of the information of interest to biologists. Expressed sequence tags (ESTs) are short stretches of sequences (usually 200-600 bp) obtained by single-pass sequencing of the 5' and 3' ends of cDNAs. This contains at least partial sequence of most mRNAs present in the various tissues used for library construction.
|
Collection of different tissues of muga silkworm larvae and total RNA extraction |
The larvae and eggs of A. assama were procured from farmers through Central Muga and Eri Research and Training Institute, Central Silk Board, Jorhat, Assam. Different tissues such as testes, ovary, brain, fatbody, epidermis, midgut, posterior and middle silkgland were dissected from day 5 of 5th instar larvae (Table 1). Dissection was carried out in ringer's solution under dissection stereomicroscope (Leica). The dissected tissues were transferred to liquid N2 and stored in -80°C. total RNA extracted from these tissues and eggs (96hrs) using the following protocol.
|
Total RNA extraction procedure |
Tissue was ground with liquid N2 in a mortar and pestle. The ground tissue was transferred into a plastic screw-cap centrifuge tube containing Trizol, approximately 10 times the volume of the tissue. Sample was incubated for 5 min. at room temperature and centrifuged at 12,000 x g at 4°C for 10 min. Supernatant was transferred into new screw- cap tube. 3ml of chloroform was added and incubated at room temperature for 2-3 min. after vortexing for 15 sec. Sample was centrifuged at 10,000 x g at 4°C for 15 min and aqueous phase was carefully transferred into new tube containing half the aqueous volume of isopropanol and 0.8 M Na-acetate. Sample was mixed by gentle inversion and incubated at room temperature for 10 min. sample was centrifuged at 10,000 x g at 4°C for 10 min. and the supernatant was discarded. Pellet was washed with 20 ml of 75% ethanol by vortexing and centrifuged at 10,000 x g at 40°C for 10 min. Pellet was briefly dried after discarding the supernatant and resupended in 250µl of DEPC treated water containing 1µl of RNase inhibiter. Total RNA thus extracted was stored at -20°C till further use for preparation of cDNA libraries and subtractive cDNA libraries.
|
Table 1: cDNA libraries prepared from different tissues and at different developmental stages of A. assama |
| Library name | Tissue type | Stage | Number of tissues isolated |
|---|
| Aaem | Embryo | 96 hours after laying | 5000 |
| Aamg | Midgut | 5th instar larvae | 20 |
| Aafb | Fatbody | 5th instar larvae | 20 |
| Aapsg | Posterior silkgland | 5th instar larvae | 50 |
| Aats | Testes | 5th instar larvae | 4000 |
| Aaov | Ovary | 5th instar larvae | 4000 |
| Aabr | Brain | 5th instar larvae | 4000 |
| Aamsg | Middle silkgland | 5th instar larvae | 50 |
| Aaep | Epidermis | 5th instar larvae | 20 |
| Aace | Compound eyes | 5th instar larvae | 100 |
|
cDNA library construction and sequencing of muga ESTs |
cDNA libraries represent the information encoded in the mRNA of a particular tissue or organism. RNA molecules are exceptionally labile and difficult to amplify in their natural form. For this reason, the information encoded by the RNA is converted into a stable DNA duplex and then is inserted into a self-replicating plasmid vector. Once the information is available in the form of a cDNA library, individual processed segments of the original genetic information can be isolated and examined with relative ease.
|
cDNA library construction |
We carried out cDNA synthesis using Stratagene ZAP-cDNA® synthesis kit following manufacturers instructions. The ZAP-cDNA® synthesis kit uses a hybrid oligo(dT) linker-primer that contains an Xho I restriction site. Messenger RNA was primed in the first strand synthesis with the linker-primer and is reverse-transcribed using StrataScriptTM reverse transcriptase and 5-methyl dCTP. The use of 5-methyl dCTP during first-strand synthesis hemimethylates the cDNA, which protects the cDNA from digestion with certain restriction endonucleases such as Xho I. Therefore, on Xho I digestion of the cDNA, only the unmethylated site within the linker-primer is cleaved.
A unique method allows construction of cDNA ready for directional insertion into Stratagene's lambda vector pBluescript® SK(-). This kit constructs a lambda library with each lot that must yield at least 2.0 x 106 pfu/5µg of control poly(A)+ RNA. After EcoRI adapters are ligated to cDNA, Xho I digestion produces vector-ready cDNA with EcoRI and Xho I compatible ends for cloning into any of the Lambda ZAP vectors.
First-strand cDNA synthesis begins when StrataScript RT, in the presence of nucleotides and buffer, finds a template and a primer. The template is mRNA and the primer is a 50-base oligonucleotide with the following sequence:
5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3'
This oligonucleotide was designed with a "GAGA" sequence to protect the Xho I restriction enzyme recognition site and an 18-base poly(dT) sequence. The restriction site allows the finished cDNA to be inserted into the pBluescript® SK(-) vector in a sense orientation (EcoR I-Xho I) with respect to the lacZ promoter. The poly(dT) region binds to the 3' poly(A) region of the mRNA template, and StrataScript RT begins to synthesize the first-strand cDNA. The nucleotide mixture for the first strand contains normal dATP, dGTP, and dTTP plus the analog 5-methyl dCTP.
During second-strand synthesis, RNase H nicks the RNA bound to the first strand cDNA to produce a multitude of fragments, which serve as primers for DNA polymerase I. DNA polymerase I "nick-translates" these RNA fragments into second-strand cDNA. The uneven termini of the double-stranded cDNA are nibbled back or filled in with cloned Pfu DNA polymerase, and EcoR I adapters are ligated to the blunt ends. The adapters have the sequence shown below.
5'-OH-AATTCGGCACGAGG-3'
3'-GCCGTGCTCCp-5'
These adapters are composed of 10- and 14-mer oligonucleotides, which are complementary to each other with an EcoR I cohesive end. The 10-mer oligonucleotide is phosphorylated, which allows it to ligate to other blunt termini available in the form of cDNA and other adapters. The 14-mer oligonucleotide is kept dephosphorylated to prevent it from ligating to other cohesive ends. After adapter ligation is complete and the ligase has been heat inactivated, the 14-mer oligonucleotide is phosphorylated to enable its ligation to the dephosphorylated vector arms.
The Xho I digestion releases the EcoR I adapter and residual linker-primer from the 3' end of the cDNA. These two fragments are separated on a drip column containing Sepharose® CL-2B gel filtration medium. Directional cDNA library was constructed by cloning of cDNA fragments into pBluescript II SK (+) vector and electroporation into E. coli strain DH10B.
|
Sequencing of Muga ESTs |
The cDNA inserts were sequenced from 5' end with RV-M primer using MegaBase 3000 (Amersham) sequencer. The sequence data was further processed by following EST processing pipeline.
|
