The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.

1. DNA & RNA
ISOLATION
Technique, Methodology and application
By Geetika Gupta
B.Sc (H) Medical Biotechnology
8th Semester
Amity Institute of Biotechnology.

2. Definition
 DNA isolation is extraction/purification of
DNA from different sources through
physical and chemical reactions.
 RNA isolation is extraction/purification of
RNA from various sources through
physical and chemical reactions.

3. DNA isolation
 It was first performed by Friedrich Miescher in 1869.
 It is used in various aspects of science e.g., Forensic sc.,
Genetic analysis, Molecular analysis, etc.
 DNA isolation from different sources require different
methodology.
 Isolation method depend on:
The source of DNA - blood, tissue, bacterial, viruses etc.
The final application – PCR, restriction, sequencing,
fingerprinting, library construction, etc
The type of DNA - genomic or plasmid.

4. DNA isolation Kit
 QIAGEN kit component:
FG 1 – Lysis buffer
FG 2 – Denaturation buffer (Isoamyl
alcohol)
FG 3 – Hydration buffer – Nuclease free
water.
 Proteinase K
 70% chilled Ethanol and Isopropanol

5. Procedure of DNA isolation
 Extraction of DNA involves 4 basic steps :
i) Preparation of cell extract from whole
blood
ii) Purification of DNA from cell extract.
iii) Concentration of DNA samples
iv) Measurement of purity of DNA
concentration

7. DNA pellet

8. Quantification
 Quantification is performed by
NanoDrop, a machine used to quantify
DNA, RNA, Protein amount in sample
and assess purity of the product.
 Earlier spectrophotometer was used to
quantify isolated DNA.

9. DNA Quantification
Sample DNA yield 260/280 260/230
C1 1154.3 1.83 1.88
C2 510.7 1.77 1.82
C3 582.8 1.78 2.12

10. DNA storage
 DNA isolation kit Qiagen and Proteinase
K is stored in 4°C.
 Whole blood (DNA sample) is stored at- -
20°C or -80°C.
 70% Ethanol and isopropanol is also
stored at 4°C.

11. DNA Check Run
 DNA check run is performed by in
0.8% of Agrose Gel Electrophoresis.
 Components of Agrose:
0.8% Agrose (0.8g Agrose in 1ml TAE)
TAE buffer

12. DNA check run result

13. Application of DNA isolation
 Used to gather evidence in a crime
investigation ( Forensic science)
 Used in genetic modification of plants
 In fingerprinting/ genetic identification
 Molecular biology investigations
 Introduction of DNA into cells of
animals and plants
 Diagnostic Purpose

15. RNA isolation
 Main purpose of RNA purification is to
isolate intact RNA for techniques like
cDNA library construction, RT-PCR, etc.
 Isolation of RNA is very sensitive and
specific process that has to be performed
with utmost precautions and care.

16. Reagents used in RNA
isolation
 RBCL – Red Blood Cell Lysis Buffer
 PBS – Phosphate Buffer saline
 TriZol – Acid Guanidinium Thiocynate
Phenol Chloroform Solution
 Chloroform
 Isopropanol
 70% Ethanol
 Nuclease free Water

17. Procedure of RNA isolation
 Homogenization
 Phase separation
 RNA precipitation
 RNA wash
 RNA solubilization

18. Homogenization
by RBCL and PBS
Phase Separation
by Chloroform
RNA Precipitation
by ice chilled Isopropanol
RNA wash
by 75% Ethanol
RNA solubilization
by Nuclease Free water

20. RNA isolation result
Sample RNA yield
(ng/ul)
260/280 260/230
C1 748.8 1.95 1.43
C2 275.3 1.91 0.68
C3 658.6 1.96 1.41
C4 1097.3 1.97 1.64

21. RNA storage
 RNA isolation reagents TriZol,
Chloroform, Isopropanol, 70%
Ethanol, Nuclease Free Water are
stored at 4°C
 Total RNA extracted stored in -20°C
 PBS and RBCL Buffer is stored at
room temperature.

22. Application of RNA isolation
Total RNA isolated can be used in:
 Northern analysis
 In-vitro translation
 Poly-A selection
 RNase protection assay
 Molecular cloning

23. Reference
 https://www.slideshare.net/AsmaAshraf7/dna-isolation-
73144905
 DNA Extraction Created by George Rice, Montana State
University
 Alaska BioPREP Virtual Textbook
 http://www.nbpgr.ernet.in/Portals/6/DMX/GENOMIC_RESO
URCES/DNA%20extraction-
Comparison%20of%20methodologies.pdf
 Rumaizah Muhamad, “RNA extraction” presentation
http://www.ukm.my/umbi/wp-
content/uploads/techrevision/RNA%20EXTRACTION.pdf
 Chomczynski P, Sacchi N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal Biochem. 1987 Apr;162(1):156-9.
 Chomczynski, P. (1993) Biotechniques 15, 532.