Chapter 3: Methods
3.10 Gene expression analysis
To confirm, equal quantity of protein was loaded on to each wells of the gel.
Membrane stripping step was performed using commercially available Stripping Buffer (Restore, Thermo Scientific, USA) for 1 hrs followed by washing with TBS-T six times in every ten mins interval. Then, the blots were re-blocked in 5% BSA for 1 hrs followed by overnight incubation with GAPDH re-probing (internal control for western experiments).
3.10 Gene expression analysis
Diluted RNA sample was quantified spectrophotometrically (Nanodrop 2000/2000c Spectrophotometers – Thermo-Scientific were used) by measuring the absorbance (A) at 260/280nm. 40 g of RNA in 1 ml gives one absorbance at 260 nm.
Therefore, the concentration of RNA in the given sample can be determined by multiplying its A260 by 40 and dilution factor. The purity of RNA can be calculated using the ratio between its absorbance at 260 and 280 nm. A ratio of absorbance at 260/280nm > 1.8 is generally considered as good quality RNA.
Real-time polymerase chain reaction (RT-PCR)
RT-PCR is a molecular biology technique used to monitor the amplification of the target DNA sequence and amplification of DNA monitored in real time as it occurs.
Principle:
RT-PCR is a method used to amplify cDNA copies of RNA (enzymatic conversion of mRNA into a single cDNA template). A specific oligodeoxynucleotide primer hybridized into mRNA and is then extended by RNA dependent DNA polymerase to create a complementary DNA copy (cDNA). In principle, PCR amplifies DNA exponentially, doubling the number of target molecules with each amplification cycle. In real-time PCR, DNA amount is measured after each cycle using fluorescent dyes yield increasing fluorescent signal, directly proportional to number of PCR product (amplicons) generated. If a specific sequence (DNA or RNA) is abundant in test sample, amplification can be observed in much earlier cycles; if amount is scarce, amplification observed only in later cycles.
Conversion of RNA to cDNA
High-capacity cDNA reverse transcription kit from Applied Biosystems was used for the conversion of RNA into cDNA.
Reagents:
10X RT buffer
25X dNTP Mix (100 mM) 10X RT Random Primers
MultiscribeTM Reverse Transcriptase enzyme Nuclease free water
Procedure:
The kit components allowed to thaw on ice and 2X cDNA conversion master mix was used. Total reaction volume of 20 µl was prepared as per Table 5.
Table 5: Preparation of cDNA concversion master mix
Component Volume/Reaction (µl)
10X RT buffer 2
25X dNTP Mix (100 mM) 0.8
10X RT Random Primers 2
MultiscribeTM Reverse Transcriptase 1
Nuclease free water 4.2
Total per reaction 10
After thawing of kit components, master mix was prepared as above-mentioned table and placed on ice and 10 µl needed for each tube (Kit capable to convert up to 2 µg RNA). 2 µg total RNA was taken and made up to the volume of 10 µl using nuclease free water. cDNA conversion master mix (10 µl) was added into tube containing 2 µg total RNA (10 µl), thus making the total reaction volume into 20 µl. The tubes were briefly centrifuged to remove air bubbles and loaded in a thermal cycler (kit specific
temperature cycles) for cDNA synthesis. The synthesized cDNA was diluted accordingly and stored at -20°C.
Steps involved in RT-PCR
There are three major steps that make up each cycle in a real-time PCR reaction. Reactions are generally run for 40 cycles.
1. Denaturation: High temperature incubation is used to ‘melt’ double-stranded DNA into single strands and loosen secondary structure of single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 92- 95°C).
2. Annealing: During annealing, complementary sequences have an opportunity to hybridize on that single strands with proper temperature, that temperature calculation based on melting temperature (Tm) of primers (5°C below the Tm of primer).
3. Extension: At 70 - 72°C, the activity of DNA polymerase is optimal, and primer extension ensued at rates of up to 100 bases per second.
Procedure
The PCR assays were carried out using SYBR green dye-based PCR. SYBR green is a commonly used fluorescent DNA binding dye that binds into double stranded DNA. Detection is monitored by measuring the increasing fluorescence throughout the cycle. 40 ng cDNA were used in PCR assays and the mastermix is prepared as per Table 6. The primer details used for assays are listed in Table 7.
Table 6: PCR master mix preparation of for sample/well SYBR® Select Master Mix (2X) 10 µl
Forward primer 0.4 µl
Reverse primer 0.4 µl
Nuclease-free water 7.2 µl
Total volume 18 µl
18 µl master mix prepared as per gene of interest and then 2 µl corresponding cDNA were added in duplicates. The plate was then sealed and briefly centrifuged at 1000 rpm to spin down the components to the well bottom and eliminated air bubbles.
Real-time polymerase chain reaction (PCR) was performed using Quant Studio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, MD, USA). The obtained data were normalized against 18s RNA (used as a reference gene), and the comparative CT (2-ΔΔCT) method was used to calculate relative quantification for mRNA expression.
CT is the cycle number at which the fluorescent signal of reaction crosses the threshold limit. Threshold limit of RT-PCR refers to the signal reflects a statistically significant increase over the calculated baseline signal. It is set to distinguish relevant amplification signal from the background.
Table 7: Primer details
Gene Forward Reverse PMID
No
Gene Accession
Number
CXCL-2 (mouse)
5'-GGATGGCTTTCATGGAAGGAG-3' 5'-TTGCTAAGCAAGGCACTGTGC-3' 22326488 NM_009140.2 CCL2
(mouse)
5'-CAGCCAGATGCAGTTAACGC-3' 5'-GCCTACTCATTGGGATCATCTTG-3' 10953027 NM_011333.2 IL-6
(mouse)
5'-ACAAGTCGGAGGCTTAATTACACAT- 3'
5'- TTGCCATTGCACAACTCTTTTC-3'
21735552 X06203 TNF-α
(mouse)
5'-AGGCTGCCCCGACTACGT -3' 5'-GACTTTCTCCTGGTATGAGATAGCAAA- 3'
21705622 NM_013693.2
IL-1β (mouse)
5'-TCGCTCAGGGTCACAAGAAA-3' 5'-CATCAGAGGCAAGGAGGAAAC -3' 21735552 NM_008361.4 COX-2
(mouse)
5'-AACCGCATTGCCTCTGAAT -3' 5'-CATGTTCCAGGAGGATGGAG -3' 22158945 NM_011198.4
iNOS (mouse)
5'-CGAAACGCTTCACTTCCAA -3' 5'- TGAGCCTATATTGCTGTGGCT -3'
22158945 BC062378.1 NRF-
2(mouse)
5’-GAGCTAGATAGTGCCCCTGG-3’ 5’-CAGGACTCACGGGAACTTCT-3’ 29162986 U20532.1 HO-1
(mouse)
5′-AAGCCGAGAATGCTGAGTTCA-3′ 5′-GCCGTGTAGATATGGTACAAGGA-3′ 25112868 BC010757.1 SOD-3
(mouse)
5'-TTCTACGGCTTGCTACTGGC-3' 5'-GCTAGGTCGAAGCTGGACTC-3' 26513461 NM_011435.3 18S
(mouse)
5'-CCCCTCGATGACTTTAGCTGAGTGT -3' 5'-CGCCGGTCCAAGAATTTCACCTCT -3' 22427817 NR_003278 CXCL-1
(human)
5’- GCGGAAAGCTTGCCTCAATC – 3’ 5’- GGTCAGTTGGATTTGTCACTGT – 3’ 25938459 BC011976.1 IL-8
(human)
5’-CTGATTTCTGCAGCTCTGTG-3’ 5’-GGGTGGAAAGGTTTGGAGTATG-3’ 20150959 BC013615.1 CXCL2
(human)
5’- TTTATTGTGGGCTTCACACG-3’ 5’- GATTTGCGCACACAGACAAC-3’ 17591792 NM_004591.3 COX-2
(human)
5’- ACAGTGTGTGGTCAACATTTCTC – 3’ 5’- TCGAAACCTCTCTGCTCTAACAC – 3’ 25938459 BC015753.1 18S
(human)
5'-GTGGAGCGATTTGTCTGGTT-3' 5'-AACGCCACTTGTCCCTCTAA-3' 25369870 NR_003286