Construction and screening of cDNA libraries-Experimentation of methods for identification of newly expressed genes under external environmental stress conditions

Summary

Materials Many organisms are excellent models for disease studies or for exploring the molecular adaptive mechanisms that enable cells and organisms to cope with and survive different stimuli. The construction of cDNA libraries and subsequent screening of target genes can lead researchers to discover new genes that have important roles in regulating and responding to certain external specific environmental stresses that may not be expressed or present in other systems. Identifying the readable frames of these new genes and further analyzing the functions of the proteins they encode can open up entirely new areas of research and allow for more scientifically designed next steps.

By Martin, this experiment is from "Environmental Genomics Experiment Guide".

Operation method

Construction and screening of cDNA libraries -Experimentation of methods for identification of newly expressed genes under external environmental stress conditions

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I. Materials

All chemical reagents were of molecular biology grade purity. Plastic and glass products used, including bottles and pipette tips were autoclaved. Gloves are required for any handling involving nucleic acids.

1. Total RNA Extraction

(1) RNase-free water. Add I mL of diethyl pyrocarbonate (DEPC) (Sigma-Aldrich) to I L of water (0.1%, V/V), stir overnight (>12 h), and autoclave. This inactivates the RNase present in the water, and the treated water is used to prepare the solutions and dissolve the RNA samples in this section.

(2) TRIzo reagent (Invitrogen).

(3) Aerosol (Fisher Scientific).

(4) Isopropyl alcohol (Fisher Scientific).

(5) 70 % ethanol with 30 mL of DEPC-treated water to 70 mL of anhydrous ethanol.

(6) Oligo (dT) cellulose (New England BioLabs, NEB). Dried Oligo (dT) cellulose was mixed with 0 -I mol/L NaOH to make a slurry and filled into a sterile column or I mL of sterilized cotton or glass-wire plugged syringe. Equilibrate the column with Sampling Buffer before spiking.

(7) Sampling buffer: lm ol/L NaCl, 2 mmol/L phosphate buffer, pH 7.2.

(8) Middle wash buffer: sample buffer + 0.3 mol,+LNaCl.

(9) elution buffer: 10 mmol/L Tris--HCl, pH7.2~7.4, I mmol/LEDTA

(10) 3 mol/L sodium acetate, pH 5.2.

(11) Ethanol.

2 cDNA library construction
3 Amplification of CDNA libraries

(1) NZY plate (24 cmX24 cm), LB-ambient penicillin broth, XLl-Blue strain, NZY agar, NZY top agar (see items 19 and 20 in 2).

(2) TSM buffer (see item 16 in 2).

(3) Chloroform (Fisher Scientific).

(4) Dimethyl satoxide (DMSO; Sigma-Aldrich).

4 Screening of cDNA library for differential expression

(1) NZY plate (24 cmX24 cm, see items 19 and 20 in 2)

(2) Hybond-14 0.45/1111 pore size nylon membrane (GE Healthcare).

(3) 20XSSC: 3 mol/L NaCl, 0. 3 mol/L sodium citrate (I L formulation: 175 g NaCl, 88 g sodium citrate); diluted with distilled water.

(4) Denaturation buffer: I.5 mol/L NaCl, 0. 5 mol/L NaOH.

(5) Neutralization buffer: I. 5 mol/L NaCl, 0. 5 mol/L Tris-HCl, pH 8.0.

(6) Rinse buffer: 0.2 mol/L Tris-HCl, p H 8. 0, 2XSSC.

(7) Whatman filter paper or chromatography paper (Fisher Scientific).

(8) dNTPs (1 0 mmol/L each of dATP, dTTP, and dGTP).

(9) Immobilized Oligo (dT) primers: 5' - dT (15) A/G/C-3, commercially available (Invitrogen; New England Biolabs).

(10) Nasin (20 U/μL, Promega)

(11) Dithiothreitol (DTT) (Sigma-Aldrich), which was prepared as a ○- I m ol/L stock solution with sterile water.

(12) A M V reverse transcriptase (10 U/μL) and 10X reverse transcription buffer (Promega).

(13) [ ^α-32P ] dCTP (3000 Ci/mol; GE Healthcare).

(14) RNaseA (60 mg/mL, Sigma-Aldrich)0

(15) Quick spin column (TE; Sephadex 025, Fine) for radiolabeled DNA purification (Roche).

(16) IX T wEl buffer: 10 mmol/L Tris-HCl, pH 8.0, I mmol/L EDTA.

(17) Hybridization buffer, modified Church's buffer: 0.25 mol/L _Na2HPO4, 0.25 mol/L _{NaH2 PO4} (pH 7. 5), lmmol/LED TA, 7 % sodium dodecyl sulfate (SDS; W/V ); or commercially available; Ultrahybe ( Ambion).

(18) Membrane washing buffer: 0. 1XSSC, 0 -1 % SDS (W/ V).

(19) Membrane rinsing buffer: 0.1 X SSC.

(20) X-ray film (Koda).

(21) TSM buffer: (see item 16 in 2).

(22) Chloroform (Fisher Scientific).

(23) XLl-Blue medium.

(24) NZY supernatant agar (see Items 19 and 20 in 2).

5 Detection of intracellular clones

(1) XLl-Blue strain.

(2) 10 mmol/L _MgSO4.

(3) ExAssist helper thumbs up the bacteriophage.

(4) Uni-ZAP X R phage storage solution.

(5) LB- Ampicillin Broth (see item 18 in 2).

(6) SOLR cells.

(7) L B Aminobenzylpenicillin Agar Plate: 15 g agar powder was added to L B broth (I L) and autoclaved. Cool to below 50°C and spread the plate, add ampicillin (lOOpg/mL) and spread the plate with proper thickness.

6 Small volume extraction of plasmids

(1) LB-aminobenzylpenicillin broth (see item 18 in 2. 2).

(2) Pre-lysis buffer: 50 mmol/L glucose, 2 5 mmol, LTris-HC1, p H 8. 0, lOmmol/LEDTA

(3) RNase A (see item 14 in 4).

(4) Alkaline lysis buffer: 0.2 mol/L NaOH, 1% S D S , which should be freshly prepared before use.

(5) Neutralization buffer: 5 m olZL potassium acetate, 30% acetic acid, 50 mm 〇 r L glucose, 25 mmol/L Tris-HCl, pH 8.0, 10 mmol/L EDTA0, 0.2 mol/L NaOH, 1% S D S , the solution should be freshly prepared before use.

(6) Isopropyl alcohol (FisherScientific)

7.70% ethanol (see item 5 of 2. 1).

7 Isolation of insertion sequences

(1) Restriction endonucleases: E co R I and Xho I (N ew EnglandBiolabs); can be adjusted according to the junction.

(2) IOX restriction endonuclease buffer; use the buffer that comes with the restriction enzymes.

(3) DNA Sampling Buffer: 0. 25% (W/V ) xylene cyanide, 0. 25% (W/V ) bromophenol blue, 50% glycerol.

(4) 1% TACE Agar Gel and 50X TACE Buffer (see items 11 and 12 in 2.2).

(5) DNA molecular quality standard (Invitrogen), depending on the size of the expressed clone fragment (from 100 bp to several kilobytes).

(6) Pipette tips with cartridges (Im L ).

(7) Sterilized cotton or glass wool.

8 Synthesis of labeled probes
9 Northern imprint

(1) 10X MOPS buffer: 200 mmol/L MOPS, 50 mmol/L acetate, 10 mmol/L EDT A , pH 7.0.

(2) I. 25% formaldehyde denaturing gel: dissolve 3.75 g of agarose in 217 m L of double-distilled water, add EB to a final concentration of Iμg / m L, and place the solution in a 60°C incubator. In a separate sterilized bottle, add 30 mL of IOX MOPS buffer and 53 mL of formaldehyde (37%, W) and place the solution in a 60°C incubator. When both solutions have equilibrated to 60°C, mix them, swirl gently to blend, being careful not to create air bubbles, and pour into a larger gelatin plate.

(3) RNA sample lysis buffer: IXMOPS buffer, 2.2 mol/L formaldehyde, 50 % formamide (V/V).

(4) 6X RNA Sampling Buffer: IXMOPS buffer, 50% formamide (V/V), 40% glycerol (V/V), add a few drops of blue bromate and xylene cyanide as tracer dye.

(5) HybondO.45 pm pore size nylon membrane (GE Healthcare).

(6) 20XSSC (see item 3 in 4), diluted with distilled water if necessary to the desired concentration.

(7) Whatman filter paper or chromatography paper (Fisher Scientific).

(8) Paper towels.

(9) Sequencing adhesive plates.

(10) Press weights.

(11) Glass or plastic pipette (5 or IOmL), used to dislodge air bubbles in the membrane transfer device.

(12) Plastic film.

(13) Fixative for Northern blotting: 0 -05 mol/L NaOH.

1 0 Detection of Northern blotting by probe

(1) Hybridization solution: Ultrahybe Hybridization Buffer or modified Church's buffer (see item 17 in 4).

(2) Radiolabeled probe (see 2. 8).

(3) Northern hybridization wash: 0.1XSSC, 0.1% SDS in distilled water.

(4) X-ray film.

(5) Northern blotting eluent: Boil double-distilled water containing 0.5% SDS, place the blotting film in, and detect the radiation signal with a Geiger counter until it falls below the background level.

II. Methods

Commercially available products designed for specific experiments (high or low copies of genes, etc.) have been developed to detect and characterize differentially expressed genes. Despite rapid technological advances, the isolation of mRNA from tissues/organs/organisms of interest to construct cDNA libraries is still the standard method for identifying new genes and proteins. There are many well-established reagents and products available for constructing a library; this chapter describes a common standard method for constructing libraries, screening positive clones and analyzing them. These experimental methods use the Uni-Zap-cDNA Synthesis Kit (Stmtagene) (5), but can be modified for use with other vectors. Some generalized phage cloning vectors include plTriplEX (Clontech), pSPORTl (Invitrgen), and ISCREEN-1 (Novagen), but are not limited to these. After library construction, differential expression screening was used to identify gene clones that were induced or repressed under the conditions tested. Here we illustrate specific experimental steps using the identification of a novel gene, expressed in hepatopancreatic tissues of sea snails and induced under hypoxic conditions, as an example. This up-regulation of expression was confirmed by Northern blotting. After the sequence determination of the imprinted-2 clone, see 3. 11 for further sequence analysis.

1 Total RNA Extraction and Isolation

(1) There are many kits available for total RNA and mRNA isolation (Ambion, Roche, Qiagen, Invitrogen, Novagen, etc.). These kits provide high purity and quality RNA, but are relatively expensive. Here we describe the traditional conventional RN A extraction method.

(2) Add Im L TRIz0I homogenate per 100 mg of tissue and add 0.2 m L' m L chloroform. Invert and mix for 15 s and leave at room temperature for 2~3 min. 4: ° C, 12 000 g■ centrifuge for 15 min.

(3) Transfer the upper aqueous phase to a new Eppendorf tube and add an equal volume of isopropanol to precipitate the RNA. leave at room temperature for 10 min.

(4) Centrifuge at 4°C for 10 min at maximum speed, remove supernatant, wash with 250 pL of 70% ethanol, and centrifuge at maximum speed for 5 min. Aspirate off ethanol, air-dry the RNA for 10 min at room temperature, and dissolve in an appropriate amount of DEPC-treated water (assuming that the amount of RNA obtained from 10 mg of tissue is IOfXg). Store at 4°C (within one week) or at 20°C (long-term storage).

(5) Determine the concentration and purity of RN A by measuring the 260 nm and 280 mn absorbance values with a UV spectrophotometer. A260/A 280 of 1.6 to 2.0 is an acceptable range (see Note 1).

(6) Purify poly (A )+ InRNA using a cellulose column incorporating Oligo (dT). total RNA is heated to 65°C, mixed 1 : 1 with Sampling Buffer (V/V), and loaded onto a cellulose column with Olig0 (dT).

(7) Rinse the column with one column bed volume of Sampling Buffer and collect the eluate in a sterile Eppendorf tube. The eluate was re-sampled, collected, and repeated twice.

(8) Rinse the column with one column bed volume of Sample Buffer and Wash Buffer, respectively, and finally elute with 1.5 times the column bed volume of eluent. As a final step, poly(A)+ InRNA is eluted by adding 0.1 times the volume of 3 mOl/L sodium acetate (p H 5-2), 2-5 times the volume of anhydrous ethanol, and leaving to settle overnight at 20°C. The procedure can be stopped here. The procedure can be stopped here.

(9) The next day, centrifuge at 13,000 g for 45 min at 4°C. Dispose of the supernatant and wash with 70% polyester ethanol (250 fxL) and centrifuge at the same speed for 10 min. The resulting precipitate is mRNA, which is air-dried and dissolved in DEPC-treated water (see Note 2). If the mRNA is not to be used immediately for experiments, store the samples at 180°C. The samples should not be used for experimental purposes.

2 cDNA library synthesis

3 Titer assay and cDNA library amplification
4 Differential screening of cDNA libraries


5 Pick-up of in vivo clones

6 Small volume extraction of plasmids
7 Isolation and extraction of insertion fragments
8 Synthesis of labeled probes
9 Northern imprint
10 Northern blotting with probes


11 Cloning Analysis

Clones with differential expression should be sequenced in both directions using standard methods (either in the laboratory or at a DNA/genome sequencing facility). Subsequent sequence analysis can be performed with commercially available software and a host of other online analysis tools. Listed below are some excellent bioinformatics software and application tools for performing cDNA and amino acid sequence analysis.
As a first step, DNA sequences should be copied to NCBI's BLAST server to compare existing and annotated sequences. The similarity of identical gene sequences or expressed sequence tags can be determined by the E-value. The E-value indicates the likelihood that each pair of sequences is similar. When the E-value of ^{-5 means that the sequence pair is not produced by error, the sequence pair is not similar}.
^{The E value -5 means that the sequence comparison is not produced by error. New or unknown genes usually cannot be paired, but some conserved regions can be identified and provide very short sequence pairs.}

(1) National Center for Biotechnology Information (NCBI)

http: //www.ncbi.nlm.nih.gov/

1. Basic Local Alignment Search Tool (BLAST): http:/ /blast, ncbi. nlm. nih.gov/Blast

BLAST first gives the residues represented by the four letters in a sequence, and then the fragments formed by the expansion of these letters. In the program used, Wastx is used to translate all the readable frames of the query nucleotide sequence into a protein sequence and then compare it with the data in the protein database. tblastx is used to translate the query nucleotide sequence and compare it with the translation results of the nucleotide sequence database. blastp is to compare the query amino acid sequence with the protein database.
2.ORF (readable frame) finder: http://ncbi.nlm. nih. gov/gorf/gorf.htmlORFfinder can identify the coding regions of all 6 readable frames in a nucleotide sequence. Selected reading frames can be directly transferred to BLAST for searching conserved regions or similar sequences (blastp).

(2) Gene translation and protein sequence identification

The identification of new gene functions is a challenge for many large-scale genome projects. While mRNA sequences contain a lot of information, proteins are usually the functional performers. To predict the function of a particular gene, the gene sequence is often translated into a predicted protein sequence to analyze homology and conserved regions with known functional proteins. In many cases, a cDNA clone usually represents a known homologue. The genes or proteins represented by these clones can often be matched to existing functional models E because these genes are likely to have been identified in other systems, organs, tissues (under various types of stress and environmental conditions) with known roles and functions. However, if the target clone does not have any known homologs, further analysis and characterization is required. Some of the software used to analyze new genes or proteins are listed below:

PredictProtein (Protein Prediction):

http: //www.embl-heidelberg.de/predictprotein<, predictprotein. html

The translated target protein sequences are searched with the PredictProtein server, and the result is a database of similar sequences and protein structure predictions.

Protein Sequence Analysis (PSA).

http:// bmerc-www.bu.edu/psa/request.htmPSA predicts the secondary and quaternary structures of proteins based on amino acid sequences.

SOSUI:

http : //sosui.proteome.bio.tuat.ac.jp/sosui_ submit,html

This tool software predicts the secondary structure of membrane proteins from the physicochemical properties (e.g., hydrophobicity and charge) of the incoming amino acid sequence.

PROSITE:

http: //www.expasy.ch/prosite

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