Isolation of RNA from animal tissues Aim: Isolation, purification and qualification of RNA from animal tissues Introduction: Ribonucleic acid (RNA) is a polymeric molecule essential in various biological role in coding, decoding, regulation, and expression of genes. RNA and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired double-strand. RNA stands for ribonucleic acid and RNA nucleotides contain three components: • A Nitrogenous Base • A Five-Carbon Sugar • A Phosphate Group The three primary types of RNA molecules are messenger RNA, transfer RNA and ribosomal RNA. 1. Messenger RNA (mRNA) plays an important role in the transcription of DNA.
Transcription is the process in protein synthesis that involves copying the genetic information contained within DNA into an RNA message 2. Transfer RNA (tRNA) plays an important role in the translation portion of protein synthesis. Its job is to translate the message within the nucleotide sequences of mRNA into specific amino acid sequences. 3. Ribosomal RNA (rRNA) is a component of cell organelles called ribosomes.
A ribosome consists of ribosomal proteins and rRNA. Ribosomal RNA is responsible for creating the peptide bonds between the amino acids in the polypeptide chain. When a termination codon is reached on the mRNA molecule, the translation process ends. The polypeptide chain is released from the tRNA molecule and the ribosome splits back into large and small subunits.
Application 1) The RNA can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, molecular cloning, and polymerase chain reaction (PCR). 2)RNA helps in amplification of DNA as the synthesis of DNA from RNA is easier. The technique to isolate, used here is a TRIzol reagent isolation, which requires a very small quantity of tissue (50 to 100 mg) and cells (5*106) and large quantities of tissue (?1 g) and cells (>107), of human, animal, plant, or bacterial origin. Principle: TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol: chloroform: isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueousphase and proteins resolves in organic phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol).
Sr no Chemical Use 1. Trizol reagent (guanidine thiocyanide) Phenol -RNase inhibitor – raptures cell membrane. 2. Chloroform -Helps in separation of three phases.
3. Iso -propanol -precipitation of RNA. 4. 70% Ethanol To remove organic solvents.
5. RNAase and DNAase free water To dilute the pellets. Procedure: Requirements: Micropipettes, Tips, Measuring cylinders, Eppendorf tubes, Beakers. Instruments: UV Transilluminator, Electrophoresis Apparatus, Incubator, Centrifuge. 1. A suspension of cell was centrifuged to obtain pallet of cell, which were dissolved in 1.
5 ml of Trizol reagent(Ambion) and kept at RT for 5 min. 2. 200 µl of chloroform was added (shaken gently 10 times) and centrifuged at 12000 rpm for 15 min at 4°C. 3.
The aqueous layer was collected in fresh Eppendorf and 500µl of isopropanol was added and left at RT for 5 min. 4. Centrifuged for 15 min at 12000 rpm.
5. The solvent was decanted and pellet were washed with 500µl of 70% ethanol. 6. Ethanol was discarded and reconstituted with DNase and RNase free water and stored in ice. 7. Isolated RNA was further quantified by nanodrop.
Evaluation: 1)Agarose gel electrophoresis: Agarose gel was prepared using 1gm agarose and 100 ml (50x) TAE- tris acetate EDTA buffer, the solution was heated at 100°C in a microwave, occasionally swirling the flask until agarose dissolved completely. It was cooled to 55-60?C and 2 µl Ethidium bromide was added and poured in gel tray with combs to create well till it solidified at RT. After the solidification of gel, isolated DNA(8µl) and RNA(5µl) were loading with bromophenol blue(5µl). The gel was run for 15 min and observed under UV radiation. 2)Quantification of RNA: Thermo scientific™ NanoDrop 2000 full-spectrum, UV-Vis spectrophotometers were used to quantify and assess purity RNA. It measures sample volumes as small as 0.
5?L. Nucleic acids absorb UV light maximally at 260nm. A260/A280 – pure RNA will exhibit an A260/A280 ratio within the range of 1.8 – 2.0 – If the RNA exhibits a ratio lower than 1.
7, this indicates protein contamination in your sample A260/A230 – properly purified RNA should exhibit an A260/A230 ratio within the range of 2 – 2.2. – If the RNA exhibits a ratio lower than 1.
7, this indicates guanidine contamination in your sampleObservation and results: • The first two wells contain DNA as single band is observed and wells containing two bands represent RNA. • Nano drop result: A260/A280 :1.26 Absorbance :0.
7 Concentration: 309.2 µg/ml Results/ Discussion: The concentration of RNA was found to be 309.2 µg/ml by using Nano drop technique. The RNA isolated was further quantified and then used for DNA synthesis and amplification.
Prepared by: Kanchan R. Sonawane 2017H1460158H