Essay, Pages 5 (1107 words)
Lysis of yeast can be attaint by homogenization trough vigorous vortexing in presence of beads (can be glass, silica or magnetic beads) or can also be accomplished by treating cells with enzymes like lyticase, zymolase etc.Techniques used in purification of RNA from yeast:Isolation of RNA is difficult from yeast because of difficulty in lysing of cell due to presence of cell wall which may also covered with capsule and yeast can also be found in spores form. After lysis, segregation of high quality RNA is also problematic to gain because of biological contamination in the sample along with presence of RNase enzyme widely disperse all over in environment.
Therefore many single and multiple steps of purification are needed for this purpose, general steps includes: lysis of cell, disruption of cellular structures creating lysate, inactivation of Cellular enzymes like RNase and at last elution of RNA from cell debris. Here we discuss timeline of techniques use in purification of RNA, quality of RNA we obtain in the end and scalability of the procedure follows: Classical eraEarlier, for analysis RNA is simply extracted with help of phenol by Kirby method (1968).
Later on scientists realizes that DNA with variety of biological component also present along with RNA and even after RNase existing in environment readily degrades RNA which leads to the discoveries of different pre and post purification techniques used for RNA.1) Cesium chloride centrifugation (CsCl) 1974:V. Glisin et.al discussed CsCl method for purification of RNA in 1974. In this cells were first washed and homogenize then CsCl was added in cellulose nitrate centrifugation tube which act as cushion.
Form top layer by adding buffer, tube were then centrifuge at 35000rpm for 12hours at 25oC.RNA is purified by centrifugation as its density is higher than DNA and most of the proteins hence settled in pallet at the bottom with the help of CsCl cushion which in the end will removed with the help of Pasteur pipet. Disadvantages of this technique are it is time consuming process, not reliable for small sample size and most importantly degradation of RNase is not always suppressed so it is difficult to make reproducible results.2) Guanidinium and chloride method 1984:Guanidinium is an de-proteinating agent which also inactivate ribonuclease enzyme rapidly, On this basis guanidinium- Cesium chloride ultracentrifugation method was introduce by Chirgwin to purify RNA from biological contaminations. In this sample were ultracentrifuge at > 32000rpm for about 12- 24 hours in presence of guanidinium which degrades protein and cesium chloride which form cushion. RNA passes through cushion and settle down at the bottom whereas DNA and other components remain above CsCl cushion.
However this classical techniques yield higher quantity of RNA but it was time taken and effort required which is also not suitable for large samples.3) Acid Guanidinium thiocyanate phenol- choloroform / trizol method 1987:It is also known as single step method for RNA isolation, introduce by Chomczynki and Sacchi. This method is the combinations of two techniques, guanidinium thiocynate and phenol- chloroform extraction methods.It is also liquid phase separation technique in which cells are first homogenized in presence of phenol and chloroform when pH is low.
This acid solution also contain guanidinium thiocynate which denature proteins along with nuclease enzymes.In this acidic solution RNA molecules remain at the top of solution and DNA remain at interphase, that’s why known as organic phase while protein settle down in to the bottom of tube known as lower organic phase.
Former methods for purification was laborious therefore they were replaced quickly by new technique. This single step method is quick which extract and purify RNA within 4 hours without ultracentrifugation providing high quality of RNA and also suitable for larger sample numbers.Scientist have different views about this technique, some have confidence in this process that it helps in extraction of good quality of RNA while other researcher avoid this method genomic contamination often present (Lewis 1997) due to presence of interphase between two layers that why this approach is often followed for recovery of total RNA by precipitation with isopropanol or ethanol.
In this era study of gene expression is on peak with the help of PCR and its different types. After reverse transcriptase ” PCR discovery (i.e. formation of mRNA to cDNA) RNA purification and amplification become easy for higher eukaryotes bur for prokaryotes and lower eukaryotes like yeast it was still hard to maintain DNA free RNA as they contain small numbers of intron in nucleic acid.4) Purification of yeast RNA suitable for modern molecular biological techniques:DNA free yeast RNA for molecular analysis is discussed by M Del Aguila et.al in 2005 that if we directly subjected sample for RT-PCR like in DNA amplification without proper purification step presences of cellular enzymes and genomic DNA present in sample produces inhibitory effect in amplification.Yeast RNA samples were first treated with DNase-1 which should be RNsae free to eliminate DNA contamination. EDTA compound was added after fee minutes in order to inactivate enzyme DNase-1 which followed by complete inactivation at 65oC for 10 minutes.But for other molecular approaches RNA was further purified with help of phenol-chloroform and followed by precipitation of RNA in presence of ice-cold ethanol.
The major problem with this technique was in RT-PCR protocol the presence of EDTA can interfere free Mg+2 concentration during reaction that effect the efficiency of RT-PCR assay and for protocol II attainment of good quality RNA, large quantity of sample required to minimize the loss during precipitation step. 5) Silica matrices:This method is based on solid phase instead of liquid- liquid purification of RNA specifically from yeast by Mutiu and Brandl in 2005 based on Nucleo-spin RNA II kit used for rapid lysis and high yield of RNA. Silica beads purification method on the base of chaotropic ion presence which quickly denature RNase.
However mammalian cells were easily extracted from this but presence of cell wall in yeast often required incubation step with lytic enzyme on the other hand if mRNA is targeted its shelf life in short so it often denature before the complete incubation period. Thus kit are now developed based on same silica matrices which did not required pre-incubation and precipitation steps for lysis of cell wall and post purification respectively.In this approach yeast RNA purify which cell walls were first broken from glass beads in presence guanidinium which quickly inactivated RNase which often result in DNA contamination. After break down of yeast cell wall with beads purification occur in two steps; first acid phenol along with chloroform was added and centrifuge that ensure the degradation of remaining RNase, DNA and protein. Secondly, direct binding with silica matrix present in kit.