SupervisorDr. Ghausiatur Reza BanuProfessor

Optimization of a protocol for extraction and quantification of total RNA from Giant Fresh Water

Prawn (Macrobrachium rosenbergii)

CandidateMd. Masud-ul-AlamRoll no: 080634

Fisheries and Marine Resource Technology Discipline

Khulna University

Khulna

Introduction

A sub-tropical climate and vast area of fresh water

suitable to prawn farming in Bangladesh

Viral diseases-a great threat to prawn farms and

hatcheries

RNA extraction and quantification for detecting and

analyzing viral disease like White Tail Disease (WTD)

Very important for adopting an optimized RNA

extraction and quantification protocol

Objectives

To develop an efficient method for RNA extraction and quantification from prawn (Macrobrachium rosenbergii)

Materials and methods

Sample collection

Berried female, larvae and post larvae (pL) of Galda

(Macrobrachium rosenbergii) from Sundarban Galda

Hatchery and Gher.

Egg, tail muscle and gill muscle taken from berried

female and juvenile.

Direct use of larvae and pL

Materials and methods contd.Protocol setup for RNA extraction

I. RNA extraction with TRIzol (Invitrogen) treating TN buffer

II. RNA extraction with AccuZolTM (Bioneer) treating TN buffer

III. RNA extraction with AccuZolTM (Bioneer) treating

DNA extraction buffer and Proteinase K

IV. RNA extraction with TRIzol (Invitrogen) treating DNA

extraction buffer

The overall procedures of four protocols were somewhat same

Materials and methods contd.RNA electrophoresis

Using TAE buffer

Using MOPS buffer

Spectrophotometric measurement of RNA

Concentration of RNA (µg/mL) = 40 x A260 of the sample

The ratio of A260 and A280 indicates the purity of RNA.

(Heptinstall, J. and Rapley, R., 2000)

Results and discussions

28S18S

A. Agarose gel electrophoresis using TAE buffer

Protocol 1 Protocol 2

Lump formation

Figure: Lump formation in protocol 2 using AccuZol

TM=Tail muscle, GM=Gill muscle, L=Larvae, E=Egg, pL= post larvae and J=Juvenile

Results and discussions contd.

28S18S

A. Agarose gel electrophoresis using TAE bufferProtocol 3 Protocol 4

B. Agarose gel electrophoresis using MOPS bufferProtocol 1 Protocol 2

Protocol 3 Protocol 4

28S18S

Results and discussions contd.Table 1: Concentration (µg/ml) and purity of RNA obtained from different protocols

Protocol no. Sample types Reading at OD 260nm

Concentration (µg/ml)

Reading at OD 280nm

Ratio (1.5-2)

1TM 1.54 61.68 0.98 1.57

GM 1.95 78.12 1.06 1.85

E 0.61 24.44 0.60 1.02

L 1.59 63.72 0.98 1.63

PL 1.68 67.04 0.98 1.71

J 1.74 69.68 0.97 1.79

2TM 1.56 62.48 0.90 1.74

GM 1.94 77.64 0.93 2.09

E 0.71 28.44 0.59 1.20

L 1.47 58.80 0.94 1.57

PL 1.51 60.48 0.86 1.76

J 1.70 68.00 0.92 1.85

3TM 1.79 71.40 1.01 1.77

GM 1.94 77.72 0.98 1.98

E 0.88 35.00 0.96 0.91

L 1.41 56.40 0.91 1.55

PL 1.52 60.80 0.87 1.74

J 1.79 71.60 0.97 1.85

4TM 1.62 64.88 0.92 1.76

GM 1.82 72.80 0.93 1.95

E 1.13 45.20 0.76 1.49

L 1.68 67.04 1.05 1.59

PL 1.86 74.24 1.12 1.66

J 1.63 65.24 0.87 1.88

Results and discussions contd.Table 2: Concentration (µg/ml) and purity of RNA obtained from different protocols

Protocol

No.

Name of Protocol Approx.

time needed

(hr)

Average conc.

(µg/ml)

1. RNA extraction with TRIzol (Invitrogen)

treating TN buffer

3.5 60.78

2. RNA extraction with AccuZolTM (Bioneer)

treating TN buffer

3 59.31

3. RNA extraction with AccuZolTM (Bioneer)

treating DNA extraction buffer and Proteinase

K

7 62.15

4. RNA extraction with TRIzol (Invitrogen)

treating DNA extraction buffer

3.5 64.90

Protocol 4 is the best considering RNA yield and time!!!

Recommendations

A separated laboratory only for working with RNA

Always using RNAse free gloves

A huge budget for chemicals

Modern equipments e.g. ice crushing machine

Further research about RNA extraction from egg of

prawn

Conclusion

Optimization of any protocol on the basis of time,

costing and quality of RNA should be kept in mind by

an ideal researcher.

In comparing the four protocols used for RNA

isolation, it was determined that protocol 4 i.e. RNA

extraction with TRIzol (Invitrogen) treating DNA

extraction buffer resulted in better purity and yield as

well as less time consuming.

Thanks to All