Question

PLEASE, I NEED TO DESCRIBE EVERY STEPS REALLY WELL. STEPS TO HANDLE AND PREPARE THE SAMPLE AND THEN, STEPS OF RNAseq.

Sequencing RNA from a single circulating tumour cell:

A research laboratory has isolated a CTC from a patient and would like a description of how you would process the sample and how you would sequence all non‐coding RNA from the cell.

a) Describe the steps involved in HOW you would prepare the RNA for RNAseq;

b) HOW RNAseq works.

Also explain WHY each step was done and consider the most appropriate method for isolating non‐coding RNA and removing rRNA from your sample.

Answer 1

a) ISOLATION OF RNA FOR RNA seq

The three major techniques extensively used for RNA extraction: organic extraction, such as phenol-Guanidine Isothiocyanate (GITC)-based solutions, silica-membrane based spin column technology, and paramagnetic particle technology. One of the most commonly used methods is the phenol-GITC-based organic extraction. But RNA samples isolated by this method are frequently contaminated with proteins, organic solvents such as phenol-chloroform, salts and ethanol. SIlica membrane based spin column is used in laboratories for isolation of RNA from patient samples. Isolation kits like Qiagen are readily available for RNA isolation and the purity is also high.

HOMOGENIZATION OF THE TISUUE

1. TRIzol reagent in a 50 ml at room temperature (RT) before taking the frozen specimen tissue.

2. As soon as the frozen tumor specimen is removed from the freezer, cut it off into small pieces while it is still frozen.

3. Immediately place the tissue into TRIzol reagent in 50 ml centrifuge tube, and homogenize using a Tissue Homogenizer for 30- 60 seconds (6000 rpm to 20,000 rpm).

4. Incubate at room temperature for 5-10 minutes after homogenization.

PHASE SEPERATION OF RNA & CELLULAR MATTER

5. Transfer the homogenate to 50 ml Centrifuge tube and centrifuge at 8000-12,000 g for 5- 10 minutes at 4ºC. The bottom pellet contains extra-cellular membranes, polysaccharides and high molecular weight DNA while the supernatant contains RNA.

6. Remove the upper layer by using a pipette. Transfer supernatant to a fresh centrifuge tube.

7. Add 0.2 ml chloroform per 1 ml Trizol reagent used. Shake the tube vigorously for 15- 30 seconds and incubate at RT for 5-10 minutes.

8. Centrifuge at 12,000 g for 15 minutes at 4ºC. Carefully remove the upper aqueous phase with the total RNA, and place in a fresh 50 ml centrifuge tube.

RNA PRECIPITAION AND WASH

9. Add 0.5 ml Isopropyl alcohol and incubate RT 10 min. Centrifuge 12,000 g for 8 min at 4ºC.

10. Wash the pellet with at least 1 ml of 75% Ethanol centrifuge 12,000 g for 5 min at 4ºC. Wash one more time with 75% Ethanol. 12. Dry the pellet at RT and re-suspend the RNA pellet in 200 –300 µl TE buffer (Make sure not to dry completely)

In SiIica membrane based spin column methods are mostly followed in kits where the protocol is readily available and easy to isolate.

b) RNA seq works using next generation sequencing techniqueto analyze the expression patterns of the transcriptome of the gene ie RNA including mRNA, tRNA and rRNA.

The first step in RNA sequencing is cDNA conversion ie converting RNAs into cDNA libraries

1. Before converting to cDNA , mRNA should be extracted, tht mRNA will represent all the transcripts of the gene

2. mRNA contains poly A tail at the end which when passed through a column of oligo dT will bind and ones without poly A tail will pass through the column. The bound mRNA can be eluted by using an elution buffer which contains glycine and HCL which will break the A-T bonds and mRNA can be isolated

These mRNA can be converted into cDNA by reverse transcriptase enzyme and other components which will then be cloned tnto plasmid vectors which upon replication produces multiple copies of itself.  

rRNA removal from total RNA can be carried out through various methods like hybridization probes that can remove the rRNA from the sample.

cDNA sequencing

Once the library is prepared, and adapters added, wecan use any sequencing platform to sequence cDNA library. Once transcript data has been produced, we can map the data to our reference genome. The alignment process can be complicated by the presence of splice variants and modifications, and the choice of reference genome used will also vary. Software packages such as STAR are used.

RNA-Seq Data Analysis

After the alignment stage, tools like Sailfish, RSEM and BitSeq will help you quantify the expression levels, while tools like MISO, which quantifies alternatively spliced genes, are available for more specialized analysis.