Materials and Methods….

CHAPTER 3

MATERIALS AND METHODS

MATERIALS

Equipments

Instruments used during the present study included- Refrigerated centrifuge

(Sigma 3K-30, Germany), DNA sequencer (3130 XL, Applied Biosystems,

USA), Refrigerated microfuge (Heraeus, Germany), C24KC Incubator

Shaker (Edison, NJ, USA), Thermocycler (ABI Perkin Elmer, USA and

Bio-Rad Research Laboratories, CA, USA), Horizontal Electrophoresis

Unit (Bio-Rad, Research Laboratories, CA, USA), UV Transilluminator

(UVP, USA), Microwave Oven (Whirlpool, India), Magnetic Stirrer

(Biosan, India ), Vortex Shaker CM101 (Remi, India), Autoclave

(Microsil, India), Rotor Plate Centrifuge (Sigma 4-15, Germany), pH Meter

(Thermo-Orion, USA), Electronic balance (Sartorius, Germany), Oven

(Yorco, India) and Water purification system (Millipore, France).

Camel populations and collection of blood samples

A total of 6 camel populations were chosen in the present investigation. For

the present study blood samples were collected from their respective

breeding tracts, comprising of Arid, Semi-arid and humid regions. The sites

of blood samples collection are shown in (figure1). About 8 ml of blood

sample was collected from jugular vein of randomly selected camel in

10ml Heparinized vacutainers and the blood was well mixed with heparin

to prevent clotting. The blood samples were transported and stored at 4oC

prior to isolation of DNA. Essentially Rajasthan, Gujarat and Madhya

Pradesh are predominantly the center of Camel rearing and breeding tracts

existing in India.

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Materials and Methods….

3.3. DNA Isolation, Storage, Quantification

3.3.1. Isolation of DNA

DNA isolation was carried out by using standard Phenol/chloroform

extraction protocol followed by ethanol precipitation from blood samples.

The RBCs were lysed with lysis buffer then mixed gently and kept in ice

for 10 minutes. The tubes were centrifuged at 12,000 rpm for 10 minutes at

4ºC in the refrigerated centrifuge. The supernatant was carefully decanted

and the pellet was redissolved and washed three times in lysis buffer. The

pellet was resuspended in 10ml digestion buffer (Sodium chloride 75 mM,

Tris-Cl 1 mM, pH 8.0 and EDTA 0.5 mM). Vortexed gently and added

20% Sodium Lauryl Sulphate (200 µl/sample) and Proteinase K (1

mg/sample) to it and incubated at 57ºC overnight in incubator. After

incubation, digested solution was obtained to which equal amount of Tris

equilibrated phenol (pH 8.0) was added. This was mixed gently by moving

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Materials and Methods….

the tubes gently in ‘8’ fashion for 10 minutes and centrifuge at 12,000 rpm

for 10 minutes at 25ºC. After centrifugation the aqueous phase and organic

phase were separated. The aqueous phase was transferred carefully by

Pasteur pipette to another Oakridge tube without disturbing the interphase.

To the aqueous phase, phenol: chloroform: isoamyl alcohol (25:24:1) was

added and mixed by moving the tubes gently in ‘8’ fashions for 10 minutes

at 25ºC to separate the aqueous phase and organic phase. Again the

aqueous phase was carefully pipetted to another tube without disturbing the

interphase. The aqueous phase was mixed with chloroform: isoamyl

alcohol (24:1). The solution was subjected to centrifugation at 12,000 rpm

for 10 minutes at 25ºC and aqueous phase was carefully transferred to glass

culture tube. The DNA was precipitated by adding 1/10 volume of sodium

acetate (3 M, pH 5.2) and 2.5 volume of aqueous phase of chilled absolute

alcohol. The DNA was spooled out into eppendorf tubes and washed twice

with 70% ethanol to remove the salt. After washing, the alcohol was

allowed to evaporate and DNA was dissolved in 500 ml Tris EDTA buffer

(Tris 10 mM, EDTA 10mM, pH8.0). Kept out Eppendorf tubes at 65ºC for

1 hour. The stock DNA was stored at -20ºC.

Estimation of DNA quantity and purity

DNA quantification was done by diluting genomic DNA in sterile

distilled water in 1:100 ratios and measuring optical density at 260nm and

280nm against a blank (sterile distilled water) using UV-Visible

spectrophotometer. The concentration of unknown double stranded DNA

samples was estimated using the formula:

Quality of Genomic DNA

Quantification of the DNA can be achieved by running the DNA samples

on 0.6 % agarose gel stained with Ethidium bromide (0.5 mg per ml) and

image was captured. Intact genomic DNA band with slight smear was

observed in wells. A smeared band towards the bottom of the gel is an

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Materials and Methods….

indication of the presence of RNA in the extract. A rough estimate of DNA

content (±50ng/l) may be obtained by comparing band intensities of the

DNA extract and the standard by eyes.

Figure: Showing quality of genomic DNA which was analyzed on 0.8

% agarose gel electrophoresis

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Materials and Methods….

3.1 Tissue Sample Collection

For RNA-Seq data generation, all the five tissues (Heart, Kidney,

testis, skin and lungs) were taken from camelus dromedarius. The fresh

tissues were steeped in RNAlater (Stabilization Reagent) immediately after

collection. RNA Stabilization reagent stabilizes RNA in tissue samples,

and then kept at −80 °C and brought to lab and processed immediately for

RNA extraction.

3.2 RNA Isolation

A typical mammalian cell contains about 10_11g of RNA of which

1-5% is poly A+ RNA. The remaining RNA is mostly rRNA (80-85% of

total) and low-molecular weight RNAs such as tRNA (15-20% of total). To

separate the heterogeneous population of mRNA from the majority of the

RNA found in the cell, affinity binding to oligo (dT) cellulose is used. This

method exploits the major characteristics of mRNA, polyadenylation, to

obtain intact, pure mRNA.

Rnase-free Glassware

Glassware used for RNA work should always be: cleaned with

detergent, thoroughly rinsed, autoclaved, then oven baked at > 210C for

atleast three hours before use. Oven- baking will both inactivate

ribonucleases and ensure that no other nucleic acids (such as plasmid

DNA) are left on the surface of the glassware. Do not use any plastic or

glassware without first eliminating possible ribonuclease contamination

from the surface of the skin. Always use proper microbiological aseptic

technique when working with RNA.

Flow Chart of Process: The following figure outlines the FastTrack@

process

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Materials and Methods….

32

Materials and Methods….

1) Preparation of lysis Buffer: Add 300µl of protein/RNase

Degrader to 15 ml of stock Buffer and use immediately.

2) Frozen tissue samples were taken out from the storage and put it in

a tared (pre-weighed), sterile, 50ml centrifuge tube and added 15 ml

Lysis buffer.

3) Sample was quickly homogenized using a homogenizer for 40 sec.

4) The cell lysate was incubated at 45°C for 15-60 minutes. If

insoluble material persists, centrifuge at 4,000x g for 5 minutes at

room temperature and transfer the supernatant to a new Eppendorf

tube.

5) 1 volume of 70% ethanol was added to cleared lysate and mixed it

well by pipetting.

6) Added 700µl of the sample into an RNeasy mini spin column

sitting in a 2 ml collection tube.

7) Centrifuged all the samples at 12,000 rpm for 15 sec. Discarded

flow-through and reused the collection tube.

8) Added 700µl RW1 buffer onto the RNeasy column, incubated

RNeasy column for 5 min, and centrifuged for 15 sec at maximum

speed. Discarded flow-through and the collection tube.

9) Transferred RNeasy column to a new 2ml collection tube.

10) Added 500μl Buffer RPE onto RNeasy column, and centrifuged for

15 sec at maximum speed to wash. Discarded flow-through and

reused the collection tube.

11) Again added 500μl Buffer RPE onto RNeasy column, and

centrifuged for 2 min at maximum speed to dry the RNeasy

membrane. Discarded flow-through and the collection tube.

12) Transferred RNeasy column into a new 1.5ml Eppendorf tube

(remove the lid) and pipetted 30μl of RNase-free water directly

onto the RNeasy membrane. Centrifuged for 1 min at maximum

speed to elute the RNA.

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Materials and Methods….

13) Transferred elute to a new Eppendorf tube and stored at -80°C in

RNase-free water.

3.3 Quantitation and quality checking of RNA

The mRNA comprises only 1- 3% of total RNA samples. It was not readily

detectable even with the most sensitive of methods. Determining the

integrity of RNA starting materials is a critical step in Transcriptome

analysis. Quantification of RNA was done using Agilent 2100 Bioanalyzer

(Agilent, Foster city, USA) which provided ng RNA/μl values. The Agilent

2100 bioanalyzer and associated RNA 6000 Nano and Pico Lab Chip kits

have become the standard in RNA quality assessment and quantitation.

Using electrophoretic separation on micro-fabricated chips, RNA samples

are separated and subsequently detected via laser induced fluorescence

detection. The bioanalyzer software generates an electropherogram and

gel-like image and displays results such as sample concentration and the so

called ribosomal ratio. The electropherogram provides a detailed visual

assessment of the quality of an RNA sample. The clear 28S and 18S rRNA

bands were indicative of intact RNA. 2100 Bioanalyzer was used with each

tissue sample and when the RNA integrity number was found to be less

than 8.5, RNA of that tissue was again isolated and further analyzed for

RNA integrity number. The RNA samples were processed further if the

RNA Integrity Number (RIN) was found to be greater than 8.5, for RNA-

Seq library preparation.

3.4 RNA-Seq library preparation and data generation

DNA library preparation was done by using Standard Illumina kit

facilitated reading both the forward and reverse template strands of each

cluster during one paired-end read. In addition to sequence information,

both reads contain long range positional information, allowing for highly

precise alignment of reads. The paired-end sequencing assay utilized a

combination of cBot (or the Cluster Station) and the paired-end module

followed by paired-end sequencing on the Genome Analyser IIx. The

unique paired-end sequencing protocol allowed us the length of the insert

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Materials and Methods….

(200–300 bp), generating high quality and alignable sequence data. A

typical paired-end run could achieve 2×76 bp reads and up to 40-60 million

reads of the transcriptome data for each of the 5 tissue samples of Camelus

dromedarius. This part of work has been outsourced.

3.5 Transcriptome data analysis

RNA-seq data analysis was done by using different software.

Transcriptome data was subjected to

Alignment against a reference genome (bovine whole genome

sequence: Btau4.0)

Identification of Molecular markers

Gene ontology

3.5.1 Quality control of RNA-Seq data

The reads may look OK in QC analyses of raw reads but some issues only

show up once the reads are aligned like low coverage, homopolymer

biases, experimental artifacts, etc. These unwanted biases can be

introduced during sample extraction process, sequencing technology,

sample preparation protocol and or may be during mapping algorithm.

SAM/BAM files usually contain information from tens to hundreds of

millions of reads. The systematic detection of such biases is a non-trivial

task that is crucial to drive appropriate downstream analyses. RNA-Seq

generated data were subjected for quality scrutiny by using SAMStat

software (Lassmann et al., 2011).

3.5.2 Alignment of RNA-seq data against reference genes

We utilized TopHat software version 1.3.2 for alignment of Transcriptome

data to cattle genome Btau 4.0 downloaded from UCSC Genome browser

(http://genome.ucsc.edu/). 3.5.3 Assembly of RNA-seq data

For assembly of transcripts, Cufflinks software version 1.0.2 was utilized.

RNA-seq read alignments in SAM/BAM format which was used as input

file for Cufflinks and it generates transcripts file.

3.5.4 Extracting gene sequencing and mapping for gene ontology terms

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Materials and Methods….

The sequences of the transcriptome were extracted using the facility of

CLCBio. All the reads were mapped with the GO IDs. The gene names

were mapped using webserver g: Profiler (http://biit.cs.ut.ee/gprofiler/) for

classification into Biological process, Cellular component and Molecular

function. REViGO web server (http://revigo.irb.hr/) was utilized for

summarizing the long lists of Gene Ontology terms associated with these

genes for removal of redundant GO terms.

3.6 Sequence Submission

All the align sequence of camel against reference sequence were extracted

and uploaded in NCBI (National Centre of Biotechnology Information).

3.7 Identification of new molecular markers

3.7.1 SNP identification

RNA-Seq data generated was mapped for each of the tissues of camel

using Illumina GAIIx with cattle genome Btu 4.0. The resultant mapped

files were then merged and utilized for heterozygous SNP calling. For SNP

detection, CLC Genomics Workbench was used which utilizes

Neighborhood Quality Standard (NQS) algorithm (Altshuler et al., 2000).

The central base quality score of ≥20 and average surrounding base quality

score of ≥15 were set to assess the quality of reads at positions for SNP

detection. We utilized the criteria of depth of coverage of ten and the minor

allele frequency of 2 out of 10 reads (minimum allele frequency of 20%)

for the identification of SNPs.

3.7.2 Microsatellite identification

For detection of STRs, novel repeat detection program PHOBOS has been

chosen for the STADEN pipeline since it implements a fast, efficient, and

highly accurate algorithm to scan molecular sequences for tandem repeats.

The minimal seed size is 5 repeat units for tandem repeats and for repeats

with longer units at least 2 exact units must be present. The Mismatch

score of -5, Indel score of -5 and recursion depth of 5 for high quality

alignment and perfection range set for 80 to 100%.

3.8 Validation of genetic marker

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Materials and Methods….

What about PCR amplification, cocktail preparation, genotyping by DNA

sequencer and data extraction……

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