The document outlines the process of RNA isolation using Trizol reagent, highlighting its effectiveness in preserving RNA integrity during tissue homogenization and the separation of RNA from DNA and proteins through chloroform addition. It describes the steps for RNA purification and subsequent cDNA synthesis, including the use of reverse transcriptase and purification methods like column purification to obtain mRNA. Additionally, the document emphasizes the significance of cDNA libraries for gene expression studies in prokaryotic systems, eliminating non-coding regions for efficient analysis.

1. T.ANANTHAKUMAR
BMS14312

2.  The isolation of RNA with high quality is a
crucial step required to perform various
molecular biology experiment.
 TRIzol Reagent is a ready-to-use reagent used
for RNA isolation from cells and tissues.
 TRIzol works by maintaining RNA integrity
during tissue homogenization, while at the
same time disrupting and breaking down cells
and cell components.

3.  Addition of chloroform, after the
centrifugation, separates the solution into
aqueous and organic phases.
 RNA remains only in the aqueous phase. After
transferring the aqueous phase, RNA can be
recovered by precipitation with isopropyl
alcohol.
 But the DNA and proteins can recover by
sequential separation after the removal of
aqueous phase

4.  Total RNA extracted by TRIzol Reagent is free
from the contamination of protein and DNA.
 This RNA can be used in Northern blot
analysis, in vitro translation, poly (A) selection,
RNase protection assay, and molecular cloning

5.  RNA will be rapidly degraded by RNases,
which are EVERYWHERE, especially on your
skin.
 To prevent RNase contamination, you must
wear gloves and use only RNase-free tips and
microcentrifuge tubes.
 All solutions have also been specially treated
with agents that deactivate RNase, like
diethylpyrocarbonate (DEPC)
 Be especially careful to keep the RNase-free
tips covered when not in use

6. REAGENTS
 Chloroform (without any additives, such as
isoamyl alcohol)
 Isopropyl alcohol
 75% Ethanol (in DEPC-treated water)
 RNase-free water or 0.5% SDS solution

7. HOMOGENIZATION
 Growth medium on the cells was discarded
and cells were washed with ice cold 1X PBS
 The monolayer was then covered with 1 ml of
TRIzol and the cells were lysed and
homogenized by repeated pipetting.

8.  The homogenized samples were incubated for
5 minutes at 15 to 30°C for the complete
dissociation of nucleoprotein complexes.
 0.2 ml (200 microliters)of chloroform per 0.75
ml of TRIZOL LS Reagent was added.
 The tubes were shaked vigorously by hand for
15 seconds and incubated them at 15 to 30°C
for 2 minutes.

10.  The samples were centrifuged for 15 minutes
at no more than 12,000 g (4°C).
 The aqueous phase was transferred to other
tubes.
 Following centrifugation, the mixture separates
into a lower red, phenolchloroform phase, an
interphase, and a colorless upper aqueous
phase.

11.  RNA remains only in the aqueous phase.
 The volume of the aqueous phase is about 70%
of the volume of TRIZOL LS Reagent used for
homogenization.

12.  The RNA was precipitated from the aqueous
phase by mixing with 3 microlitre of glycogen
and 500 microlitre of isopropyl alcohol.
 The mixture was centrifuged for 30 minutes at
12,000 × g (2 to 8°C).
 ( The RNA precipitate forms a gel-like pellet on
the side of the tube at bottom).

13.  The supernatant was removed.
 The RNA pellet was washed once with 75%
ethanol, adding 900 microlitre of 75% ethanol
per 0.75 ml of TRIZOL LS Reagent used for the
initial homogenization.
 The sample were inverted and mixed and
centrifuged at 12,000 rpm for 30 minutes at 4
degree.

14.  RNA was dissolved in RNase-free water (or
0.5% SDS solution) by passing the solution
through the pipette tip for a few times, and
incubating for 10 minutes at 55 to 60°C.

15.  cDNA is created from a mature mRNA from a
eukaryotic cell with the use of an enzyme
known as reverse transcriptase.
 In eukaryotes, a poly-(A) tail (consisting of a
long sequence of adenine nucleotides)
distinguishes mRNA from tRNA and rRNA
and can therefore be used as a primer site for
reverse transcription

16.  Firstly, the mRNA is obtained and purified
from the rest of the RNAs.
 Several methods exist for purifying RNA such
as trizol extraction and column purification .
 Column purification is done by using
oligomeric dT nucleotide coated resins where
only the mRNA having the poly-A tail will
bind.

17.  The rest of the RNAs are eluted out.
 The mRNA is eluted by using eluting buffer
and some heat to separate the mRNA strands
from oligo-dT
 Purification can be performed by binding
mRNAs on a solid matrix to which short
strings of thymidylate residues are attached
(oligo dT matrix).
 The mRNAs are removed again by washing in
a low salt buffer.

19.  Once mRNA is purified, oligo-dT is tagged as a
complementary primer which binds to the
poly-A tail providing a free 3'-OH end that can
be extended by reverse transcriptase to create
the complementary DNA strand.
 Now, the mRNA is removed by using an
RNAse enzyme leaving a single stranded
cDNA (sscDNA).
 This sscDNA is converted into a double
stranded DNA with the help of DNA
polymerase.

20.  However, for DNA polymerase to synthesize a
complementary strand a free 3'-OH end is
needed.
 This is provided by the sscDNA itself by
generating a hair pin loop at the 3' end by
coiling on it.
 The polymerase extends the 3'-OH end and
later the loop at 3' end is opened by the
scissoring action of S1 nuclease.

23.  cDNA libraries are commonly used when
reproducing eukaryotic genomes, as the amount of
information is reduced to remove the large
numbers of non-coding regions from the library.
 cDNA libraries are used to express eukaryotic
genes in prokaryotes.
 Prokaryotes do not have introns in their DNA and
therefore do not possess any enzymes that can cut
it out during transcription process.
 cDNA do not have introns and therefore can be
expressed in prokaryotic cells.

24.  Also, it is useful for subsequently isolating the
gene that codes for that mRNA.
 Discovery of novel genes.
 Clonning of full length cDNA molecules for in
vitro study of gene function.
 Study of the repertoire if mRNAs expressed in
different cells or tissues.