Detection of cytosolic tRNA in mammal by Northern blot analysis

Out of entire cascade of technologies and strategies, Northern blot assay remains the most preferential approach for immediate and accurate evaluation of expressed RNA species. However, an abundance of tRNAs species under physiological conditions compared to other small RNAs makes it difficult to accurately evaluate their transcriptional alterations through traditional Northern blot assay. Here, we describe an efficient protocol for detecting subtle alterations in tRNA species in mammals by a modified Northern blot assay. This report also compares the chemical versus UV-based crosslinking of tRNA species to the surface of solid supports.


INTRODUCTION
Transfer RNAs (tRNAs) are small adapter molecules of approximately 70 nucleotides in length that translate codons in mRNA into their cognate amino acids in a polypeptide chain. Protein biosynthesis process requires tRNA molecules to actively participate in interaction with several proteins such as tRNA modifying enzymes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, and a repertoire of ribosomal RNAs, proteins and mRNA, therefore, estimation of alterations in tRNA profile has become quintessential. Last decade has fuelled the development of powerful technologies dedicated for profiling of steady state accumulation of specific small RNA species.
Northern blot assay was developed in the year 1977 (Alwine et al., 1997) and since then, it has revolutionized the RNA biochemistry field. Past decades have experienced spectacular progress in the molecular biology through the advent of powerful technologies developed dedicated for quantifying and manipulating RNA species expressed in the cell (Gesteland et al., 2006). However, Northern blot forms the basic fundamental, quick and clean, easy-to-go technique behind more sophisticated and costly RNAbased analysis. The tRNA Northern blot procedure has been adapted from Wei et al., (2013).

PROTOCOL
The protocol takes three days to complete and further two to three days to expose and develop the autoradiography film.
Denaturing urea polyacrylamide gel electrophoresis 1. Perform total RNA extraction from desired source (tissue, monolayer cells, suspension cells) using commercial reagents e.g., TRIZOL reagent RNA extraction protocol by following manufacturer's instructions and recommendations 2. In order to assess the quality of RNA load 2 μg of total RNA on 0.8% EtBr-agarose gel and run the horizantal electrophoretic unit at 50 V in 1x TBE buffer until RNA species are resolved (15 ˜20 min).
Identify the distinct bands corresponding to 28S, 18S, and small RNA species such as 5.8S and tRNAs 3. For the RNA fractionation step, use large vertical electrophoretic apparatus (Plate size 20 cm x 16 cm, comb size 1 mm). Proceed with the assembly of clean glass plates together in casting frame by following manufacturer's description. Larger gels are used in order to enhance resolution and separate bands with a difference of single nucleotide 4. Prepare 60 mL of 15 % acrylamide/bisacrylamide (29:1) casting solution in 1x TBE, 8M ultra pure urea and fill the volume by DEPC treated deionized water. Dissolve the components by swirling the flask and sterilize the gel casting solution by passing through a 0.4 μM filter with the help of 50 mL syringe. Immediately, before pouring add 590 μL of 10 % APS and 24 μL of TEMED. Pour the gel immediately in between the vertical plates in gel casting frame and avoid introduction of air bubbles. Carefully, insert the comb and allow the gel to polymerize for 1 hour at room temperature before use

RNA sample preparation
1. Aliquot 20-30 μg of total RNA for each sample and add an equal volume of 2x acrylamide blue loading dye (2x LD) 2. Denature the RNA sample by heating at 80ºC for 10 min and chill on ice until loading Fractionation by electrophoresis 1. Gently remove the comb and dismount the gel from casting frame. Carefully assemble the gel in the electrophoretic cell, following manufacturer's instructions 2. Fill the lower and upper chamber with 1x TBE running buffer. The lower and upper chamber should be filled with running buffer to the same level 3. Attach the lid and connect the cables to the high voltage power supply. Apply the constant voltage ˜450 V and pre-run the electrophoretic unit for 1 hour 4. Rinse the well pockets by squirting 1x TBE buffer through a sterile syringe 5. Carefully load samples from the bottom of the well. Assemble the apparatus and apply constant voltage ˜100 V for 30 min, followed by an increase in voltage to ˜200 V and run for 4-6 hours 6. When the bromophenol blue dye front reached the end of the gel, carefully dismantle the assembly and de-clamp the gel. Cut the gel into pieces as per your requirement 7. In order to visualize fractionated RNA on the gel, incubate the gel slice in 1x TBE buffer containing sufficient ethyl bromide, at room temperature for 20 min and place on the slow rocker. Additional RNA sample should be loaded as a control for fractionation on gel. Destain the gel in 1x TBE for 20 min and visualize fractionated RNA by UV irradiation based gel imaging system

Gel blotting
1. Measure the gel and carefully cut 6 pieces of Whatman blotting paper and nylon membrane of equivalent size. Equilibrate the gel pieces, blotting papers and nylon membrane in 1x TBE 2. Clean the surface of semi-dry transfer apparatus with RNase Wiper and place 3 pieces of soaked blotting paper on the surface of apparatus. Carefully remove air bubbles and excess TBE buffer by means of a sterile roller and dab the surface from the edges 3. Place the buffer-equilibrated nylon membrane on the blotting papers and remove the excess TBE buffer from the edges. Place the gel containing fractionated RNA on the nylon membrane and remove the excess TBE buffer 4. Sandwich the nylon membrane and gel by covering with more 3 soaked blotting papers and carefully roll out the air bubbles and excess TBE buffer using a roller 5. Carefully check the orientation of electrodes and assemble the transfer apparatus 6. Transfer RNA from the gel to the nylon membrane at constant 10 V for 2.5 hours The schematic of Northern blotting is shown in Figure 1.

5.
Assemble the membrane/film sandwich using exposure cassette and intensifier screen following manufacturer's recommendations in dark room and allow the film to expose for almost 2 days and develop the exposed film using film developer protocol Add DEPC-treated water to make final volume up to 1 L and store room temperature

RESULTS
We followed the procedure described in the text and estimated the cytosolic methionine initiator tRNA and cytosolic glycine tRNA expression from samples of mammalian origin. We also compared the tRNA crosslinking efficiency to a solid support using UV crosslinker strategy and carbodiimide based chemical cross linking technology. RNA was isolated from HEK293T monolayer cells by using TRIZOL reagent method and integrity of RNA was assessed by visualizing discrete bands on an agarose gel and also by estimating OD260/280 (˜2) and OD260/230 (˜1.9) ratio on NanoDrop.
RNA samples (30 μg) for cytosolic methionine initiator tRNA were run in duplicates. One replicate was blotted on neutral nylon membrane and chemically cross linked by EDC whereas, another replicate was blotted on negatively charged nylon membrane and crossed by UV.
The expression of cytosolic methionine initiator tRNA was detected and confirmed through both methods ( Figure 2). However, the chemical crosslinking with EDC exhibits higher sensitivity and enhanced specificity than traditional UV crosslinking. Therefore, utilization of EDE based chemical crosslinking could largely benefit small sized and less abundant species of tRNA molecules.
Gly-tRNA iMet-tRNA iMet-tRNA Chemical Crosslinking UV crosslinking Figure 2: Northern blot for detection of methionine initiator tRNA. Equal amounts (30 μg) of RNA sample was resolved on a denaturing urea polyacrylamide gel and subjected to either EDC based chemical crosslinking or traditional UV based crosslinking after electro-blotting to the nylon membrane

DISCUSSION
The variation of EDC-based chemical crosslinking was developed and used for first time by Giangrande et al. to detect weakly expressed microRNA from Drosophilla melanogaster whole embryos (Laneve and Giangrande, 2014). Here we, utilize the similar strategy to detect the population of small RNA species such as cytosolic methionine initiator tRNA and glycine tRNA isolated from HEK293T cells. It is clear from our results that EDC-based chemical crosslinking method exhibits increased sensitivity with an average of around 50-fold improvement of target detection.
One of the critical determinants of RNA-based technologies is the maintenance of the integrity of RNA.
The ubiquitous presence of RNases limits the process of RNA manipulations because of enhanced chances of RNA degradation right after the extraction until gel loading. Therefore, all equipment, glassware/ plastic ware should be thoroughly wiped with RNase-based surface decontaminant and followed by rinsing with DEPC-treated water. All the solutions should be prepared by using DEPCtreated water as a solvent.
Residual DEPC or DEPC by-products have been known to interfere in several enzymatic reactions and is also known to modify RNA (carboxymethylation), therefore, DEPC should be inactivated and removed by autoclaving at 121ºC for at least 30 to 40 min. Also, 10x TBE should not be prepared directly in active DEPC containing water. The presence of nucleophile amine groups of TRIS would hydrolyse and inactivate DEPC, therefore treatment against RNAse would not be complete. It is highly recommended to prepare 10x TBE in already autoclaved DEPC-treated water, followed by 2nd round of autoclaving.
The EDC mediated chemical crosslinking technology is based on the formation of covalent bond between RNA 5'monophosphate group and primary amines on nylon membrane. The involvement of only 5' head of RNA in crosslinking leaves the rest of RNA free for hybridization, which enhances the sensitivity of assay.
The sharpness of the band depends on several parameters. However, small sample volume (2-5 μL), thin gels, loading of sample from bottom of the well and removal of urea cushions from the well pockets ensure sharper and better resolved the RNA bands. It is also recommended to initially run at lower voltage (100 V for 30 min) or cast 4% stacking gel over separating gel, in order to enhance the sharpness of bands.