rna sequencing

rna sequencing

Transcriptome Analysis Takes Predictive Breeding a Step Ahead
With the advent of genetic engineering, there has been a drastic change in the agricultural techniques. Gone are those days when man used to depend solely on nature for rainfall, soil fertility etc. Modern-man can toy with the genetic sequence and identify the part of genome which is beneficial for him.

An attempt to improve the crop varieties began almost simultaneously with the agricultural practices. Around ten thousand years ago, man started to alter the genetic make up of crops without his knowledge. The conventional plant breeding practices began when early farmers selected the plant with desirable traits and saved its seeds for the next generation. Later with the knowledge of genetics, plant breeders were able to select the crops with desirable traits like faster growth, pest and disease resistance, sweeter fruits, larger seeds etc. Two plants having two different desirable characters were cross-pollinated artificially to obtain an offspring containing a mixture of these characters (hybrid variety). As a net result of these techniques new varieties of crops have developed which are significantly different from their wild relatives. Conventional breeding techniques largely depend on selection of superior varieties of crops and incorporation of desirable traits into future generations.

Genetic engineering has brought quantum change from conventional breeding practices and has introduced combination of genes which do not exist naturally. Sequencing of genomes of various plants has now become feasible with the faster progression in genetic engineering. Predictive breeding is an advanced technique which comes up with a promise of identifying the beneficial genes out of the entire genome. Genetic markers like SNPs (single nucleotide polymorphism) or minisatellites are used to construct linkage maps. The linkage maps help to identify the traits which are inherited together. The knowledge of genome sequence along with the linkage maps helps to associate the beneficial traits of the plant with specific parts of genome sequence.

Complicated genomes of polyploid crops pose a serious problem to predictive breeding. A cell of a polyploidy organism has more than two sets of homologous chromosomes and each set contains all the genes responsible for the biological traits of that organism. Professor Ian Bancroft took this as a challenge and identified the sequence of rapeseed (Brassica napus). This crop has been found to originate from two related species that is Brassica rapa and Brassica oleracea. Rapeseed (Brassica napus) was declared as a third leading source of vegetable oil in the world in the year 2000. Canola is the name given to Canadian oil that is extracted from certain varieties of Rapeseed. This oil is a rich source of essential fatty acids (omega-6 and omega-3 fatty acids in the ratio of 2:1). Apart from these it is also an important source of Biodiesel which can power motor vehicles. This oil is generally combined with fossil-fuel diesel in ratios varying from 2% to 20% of biodiesel. This does not damage the engine. Isn't it an eco-friendly plant?

Sequencing the DNA of this plant directly was difficult because of its complicated genome. Hence they adopted a different strategy. Transcriptomes were sequenced from young juvenile leaves. Transcriptome is a set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA. It represents a small percentage of the genome. The sequence of a transcriptome mirrors the sequence of the DNA from which it was transcribed. This study gives an idea about all the genes which get expressed into proteins in that tissue. Sequence of transcriptome will act like a raw material for scientists struggling to locate the human friendly genes within the entire genome of the polyploid plants. Based on this improved varieties of seeds can be produced.

A large number of crops like potato, wheat, oat, sugarcane, cotton etc are polyploid and few crops amongst them are staple crops in certain parts of the world. Transcriptome sequencing will help the scientists to identify the desirable genes even in the absence of complete genome sequence. This new technique will make predictive breeding of crops with complicated genomes feasible.

Article Source: rna sequencing

rna synthesis

rna synthesis
 This procedure uses denatured RNase T1 to digest RNA. RNase T1 cleaves single-stranded RNA residues of guanosine 3  '. Partial digestion of 3  'or 5  like' RNA with this enzyme thus produces a scale of residue G.

 The 1X rna synthesis Buffer used in the procedure containing 7 M urea denaturing contribute to RNA secondary structure. To perform the procedure diluted the RNA ends at least five times in buffer sequencing. The sample is denatured at 50 ° C, RNA and heatedthen incubated with RNase T1 at room temperature.

rna synthesis 
In the process, two different concentrations of RNase T1 can be used. Tube # 1 contains RNase and represents a negative control. Non-Bands in length in the tube are fission products within the RNA sample. These bands will also be present in samples of nuclease treatment, should be ignored in its analysis. T1 Tubes # 2 and # 3 with two different concentrations of RNase. Normally, at least one of the reactionscreate a ladder with digestion by each of the guanosines in the rna synthesis . Further enzyme dilution may be necessary to achieve the optimum scale digestion.