Structural Biochemistry/Nucleic Acid/RNA/Short RNAs Sequencing

Introduction

The short RNAs includes different classes of molecules like small nuclear RNA, micro RNA, and transfer RNA and etc. Many functional RNAs are less characterized as short RNA because short RNAs are found at the promoter and 3' termini of gene sequeces . It also involves in paramutation, is a reciprocal action between two alleles at locus, inducing an intermolecular change in the other allele. The short RNAs can be studied in research field for describing and analyzing the stratigies and process to develop short RNA species with single-molecule sequencing (SMS) and its efficiency in many laboratory research. The Short RNA is derived from the final product of functional precursor RNA species in the cell. One of the examples of the short RNA species in the cell can be found in gene splicing in DNA sequence and 3' end RNA processing for synthesizing grown-long mRNAs . However, many classes related to functional RNAs that are not in protein coding are most likely from precursors which are longer than grown up long mRNAs. To fractionate RNA into species some methods are being used to study the various functions of the short RNAs. For example, Dr. Philipp Kapranov, and his researchers uses the method of Helicos single-molecule sequence to study the details of short RNAs in the cell .

RNA Isolation for short RNA

To proceed the isolation of RNA, sRNA gets purified from total RNA (cultured cells) with mirVana miRNA Isolation Kit and miRNeasy Mini Kit, which also is known as Qiagen. The RNA Kit is used for large amount of sRNAs (from cultured cell) preparation . Through this method of RNA/DNA kit, sRNA of specific fractions gets purified with Elution and Electrophoresis method of TBE-Urea polyacrylamide gel. Electrophoresis is action of spreadout particles in fuild due to spartially uniform electric field throughout the cell structures.

Difficulties of Analysing short RNAs

Studying of the short RNAs is very challenging among scientists and researchers. There are some difficult problems that challenges researchers who are studying about short RNAs: 1) Specific Isolation of sRNA must be successful with desired length range. 2) There's lack of interest about sRNAs with molecular handles ike 3' PolyA Tail of the mRNAs which is used for converting itself into cDNAs. 3) sRNA is sometimes too short for efficient and successful conversion into cDNA with hexamers. 4) Some specific sRNAs have modification at gene sequence of their 5' and 3' ends that interrupt the reading with ssubsequent molecular manipulation; therefore, often few sRNAs cannot be detected by some methods that a lot researchers are using. 5) Some classes of sRNAs have firm 2' structures that blocks itself from being detected by enzymatic methods which are often conducted by researchers under nondenaturing condition. 6) Certain sRNAs such as miRNAs have short lengths that are not capable of being used for efficient mapping to complex genomes; thus, discovering sRNAs and its specific functions are very challenging for researchers to study and conduct experiments related to sRNAs. 7) Many methods depend on using ligation process and PCR amplication that can misrepresent the composition and quantification of the RNA species in the cell structures. Moreover, 8) Fraction of sRNA can be dominated with very highly presence of RNA classes like snRNA, rRNA, and sno RNAs (These will require a lot of complexity for completing characterization of the sRNA population in the cell. The most challenging part of studying sRNAs happens to deal with the physical structure in the cell: short length of sRNA makes researchers difficult to conduct an experiement .

Isolation of sRNA Fraction

Using diverse methods, sRNA fraction can be detected and isolated with using either miRNeasy, or DNA/RNA Kit (Qiagen) . If wanted, sRNA Isolation can be conducted in specific size range by using TBE-Urea denaturing polyacrylamide gel electrophoresis as another substitutive method for both researchers and scientists who are studying for sRNAs. One of the method of studying sRNAs deals with Tailing of RNAs with 3' PolyC: RNA is mixed with water in PCR amplication tube and it is used with protocol; then, sRNA gets incubated for 2 minutes at 85 degress Celcius in a PCR Machine and put into ice 2 minutes at least. After its incubation, reagents are added to the solution with E. coli Poly A polymerase buffer and mixed. SRNA gets extracted twice with addition of phenol or chloroform or isoamly for few seconds; then, purification with addition of 100% EtOH (Ethanol) for half an hour with minus 80 degrees Celcius. Centrifuge at 4 degrees of Celcius and leave it for 30 minutes. Then, washed with 70% EtOH and finally resuspend with water .

Preparation of cDNA Sequence

The cDNA can be sequence from the 3' end with poly-A Polymerase in the method of terminal transferase (TdT) and be blocked at the other 3' end .thus, the cDNA can be bind to the oligo-dT present at the surface of the cell. This method is common for protocol and for poly A protocol. Some amounts of cDNA are analyzed under experiement with use of regular NanoDrop if the expected concentration of cDNA is at certain range between 5-10 ng. Tailing part of cDNA is followed: 1) cRNA is prepared to be tailed in water and depending on its volume, its time spending in the Incubation varies and amount of buffer can be differed. After Incubation, certain micro-measured amount of TdT buffer is added into the cDNA soltuion; The cDNA gets incubated again in the PCR Amplication machine at 37 degrees of Celcius for an hour. Incubated cDNA sample gets heat up to 95 degrees of Celcius for 5 minutes. Later, TdT Buffer is added and incubation is repeated to get final sequence of cDNA and measure the concentraion of tailed cDNA in the sample.

Reference

1. http://www.ncbi.nlm.nih.gov/pubmed/22144202
2. http://en.wikipedia.org/wiki/Electrophoresis