实验报告(RNA).docx

2011030014 宋予晴

Title:
Isolation of Total RNA from Animal Tissues and Gel Electrophoresis

Background:
Objectives:
• Master the principle, methods of RNA isolation and detection;
• Learn some related techniques for handling RNA.
Principle:
Each mammalian cell contains about 10pg of RNA, of which 80-85% is ribosomal RNA (rRNA) (28S, 18S and 5S varietes), 10-15% is made up of smaller species, such as transfer RNA(tRNA) and 1-5% is messenger RNA(mRNA).
Most mRNAs have a long tail of adenine(A)-containing nucleotides (PloyA tail) in 3’ terminus. Therefore, mRNAs can be isolated from other cellular RNAs by affinity chromatography on oligo (dT) cellulose. Generally, the total RNA isolated can also be used in Northern blotting.
RNA Isolation:
RNA Isolation needs the following step:
1. Grind or homogenize tissue samples
2. Add protein detergent to separate RNA from nucleoproteins and release RNA.
3. Inhibit endogenous and exogenous RNase activity.
4. Isolate RNA from DNAs, proteins and other cell components.
Several methods for RNA isolution:
1. Phenol method: Use SDS to denature proteins and inhibit RNase activity. Perform phenol/chloroform extraction to get rid of proteins, polysaccharides and pigments. The RNAs are then precipitated with NaAc and ethanol.
2. Guanidinium method: Use guanidine isothiocyanate or guanidine hydrochloride and b-mercaptoethanol to denature proteins and inhibit RNase activity. Perform phenol/chloroform extraction and then precipitate.
3. Lithium chloride method: At certain pH, Li can specifically precipitate RNA, but small RNA will be likely lost, and the remained Li also will inhibit mRNA activity.
In our experiment, we used TRIzol Reagent from Invitrogen company. The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, is an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi(1). During sample homogenization or lysis, TRIzol Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation will separate the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol.
RNA Detection:
RNA is mainly detected by agarose gel electrophoresis, including non-denaturing and denaturing electrophoresis. The most commonly used method is denaturing electrophoresis by adding formaldehyde to agarose gel.
In our experiment, we used the non-denaturing electrophoresis because we needn’t do the Northern blot analysis.

Materials & Equipment:
Materials:
• fresh tissues from mouse
• Invitrogen TRIzol Reagents
• liquid nitrogen
• chloroform
• isopropyl alcohol
• 75% ethanol
• 10ⅹgel electrophoresis buffer:
n Morpholinopropanesulphonic acid( MOPS)（pH 7.0） 200mmol/L
n NaAC 50mmol/L
n EDTA (pH 8.0) 10mmol/L
n Prepare with DEPC treated water , adjust pH=7.0 with NaOH, filter to get rid of bacterial, and preserve in dark .
• 10ⅹ loading buffer:
n 50％(V/V） glycerol(dilute with DEPC treated water)
n 10mmol/L EDTA(pH8.0)
n 0.25％(m/V) Bromophenol blue
n 0.25％(m/V) Xylene cyanol FF
Equipment:
gel electrophoresis system
CCD imaging instrument

Procedure:
RNA Isolation
1. Weigh 100mg of fresh tissues (kidney for our group), freeze in liquid nitrogen.
2. Grind the samples in pre-chilled mortars with liquid nitrogen, then transfer the powders to 2ml or 1.5ml microcentrifuge tube.
3. Add 1 ml of TRIzol Reagent to samples and mix it. Incubate the homogenized samples for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.
4. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent . Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at room temperature for 2 to 3 minutes.
5. Centrifuge the samples at 12,000×g for 15 minutes at 4ºC.
6. Transfer the aqueous phase to a fresh tube. Precipitate the RNA from the aqueous phase by mixing with 0.5ml of isopropyl alcohol, mix well. Incubate samples at 15 to 30ºC for 10 minutes.
7. Centrifuge at 12,000 × g for 10 minutes at 4°C, discard the supernatant, and keep the pellet.
8. Wash the RNA pellet once with 1ml 75% ethanol. Centrifuge at 7,500 × g for 5 minutes at 4°C.
9. Briefly dry the RNA pellet. Dissolve RNA in 50ml RNase-free water
RNA Detection
1. Prepare gels(1.2%): prepare 1.2% gels with 1ⅹgel electrophoresis buffer, settle for 30min .
2. Prepare samples: mix samples with 10ⅹloading buffer at a ratio of 9:1 in microcentrifuge tube, quickly place it on ice for 5min, centrifuge for several seconds. The loading amount is usually 2-30 m g. For this experiment, 10 m g is loaded.
3. Before loading, the gels should pre run for 5min, then load the samples, and run at the voltage of 5V/cm, for 1-1.5h.
4. End the electrophoresis when bromophenol blue reaches to 4/5 of the gel. Dye the gel in EB solution for about 30min.
5. Observe the result under UV light.

Result & Analysis:
5S
18S
28S

In the graph, we can see most of the RNA has 3 bands: the 28S on the top, the 18S in the middle and the 5S at the bottom. However, the fat lane has nothing, it might because the RNA was thrown out after the centrifuge. The stomach and the kidney lanes were too bright to see the band clearly which might be caused by loading too much RNA when doing the electrophoresis. The spleen lane also has many other bands of RNA.