Isolating mRNA and protein together from biological samples can be a challenge for researchers, especially when the samples contain very small numbers of cells to start with. In a Benchmark article in the current issue of BioTechniques, a team of researchers from University Hospital in Copenhagen describe a new approach for the simultaneous isolation of mRNA and protein from biological samples. The authors use oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads along with an optimized reaction buffer to isolate the mRNA, while the protein content is retained in a native state that is suitable for proteomic analyses. Validated using both neonatal rat ovaries and human granulosa cells, this new technique should be a welcome addition for those researchers working with very precious biological samples.
Recombinant adeno-associated virus (rAAV)-based vectors have proven to be efficient tools for targeted genome engineering. Although effective, generating the viral targeting vectors can be a time-consuming process for investigators at the bench. In the current issue of BioTechniques, a team of researchers from Aarhus University introduce a new method for generating rAAV vectors based on a Golden-Gate cloning strategy. The authors demonstrated the ability to generate both gene knockout and single nucleotide knock-in vectors using the new approach. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as assembly modules to confer antibiotic resistance to the targeting vector. In addition, the pGolden-AAV vector was designed to allow assembly of rAAV-based targeting vectors in a single step. This new toolkit for generating rAAV vectors should go a long way in enhancing the utility and impact of rAAV-based genome engineering applications in the future.
To date, genome-wide RNA analyses have given short shrift to 3′ termini. This is understandable given the difficulty of sequencing poly(A) tails, but also unfortunate since 3′ termini help dictate mRNA regulation and fate. TAIL-seq addresses this gap by genome-wide measurement of poly(A) tails. In the procedure, small noncoding RNAs are size fractionated away, followed by 3′ adaptor ligation, fragmentation, addition of the 5′ adaptor, RT-PCR, and Illumina sequencing. The reads comprise a 51-nucleotide sequence from the 5′ end (for mapping) and 251 nucleotides from the 3′ end (for characterizing the terminus). Key to sequencing the homopolymeric runs is including degenerate bases in the 3′ adaptor and a new algorithm that corrects for accumulation of the T signal while reverse sequencing the poly(A) tail. TAIL-seq of mammalian cells showed shorter poly(A) tails than anticipated, as well as uridylation/guanylation.
H. Chang et al. TAIL-seq: Genome-wide determination of poly(A) tail length and 3’ end modifications. Mol Cell. [Epub ahead of print, February 27, 2014; doi:10.1016/j.molcel.2014.02.007].IMPROVING FILTER-AIDED SAMPLE PREPARATION
To ready samples for mass spectrometry, an alternative to acetone precipitation is filter-aided sample preparation (FASP), which uses buffer exchanges in spin filter units to remove denaturants and other contaminants. FASP's weakness is sample loss, which is addressed in a new procedure called enhanced FASP. The first improvement is overnight Tween-20 passivation of the filter unit to reduce protein adsorption to the surface. Second, the digestion buffer substitutes deoxycholic acid for urea, improving trypsin efficiency. This effect holds even in hydrophobic regions of proteins, so enhanced FASP may improve identification of membrane proteins. Another variant of FASP, express FASP, offers one-step reduction and alkylation. In this case, the alkylating agent is 4-vinylpyridine, which is compatible with the reductant tris(2-carboxyethyl)phosphine (TCEP), allowing a buffer exchange step to be bypassed. Express FASP, like the enhanced version, improves sensitivity, recovery, and coverage in proteomic analyses.
J. Erde et al. Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J Proteome Res. [Epub ahead of print, March 6, 2014; doi:10.1021/pr4010019].