A: Yes. Bio Rad precast TBE gels (Cat. No. 4566033) are fully compatible with the EZ QC™ mRNA Poly(A) Tail Length Assay.
A: ~0.1 pmol of poly(A)-tailed mRNA can work under the standard reaction conditions. However, for easier detection and a more accurate evaluation of the poly(A) tail size distribution it is recommended to use ≥2.5 pmol of input mRNA. Note: these values could vary based on the efficiency of tailing, gel exposure time, quality of gel loading and other factors.
A: The tolerance of the assay is such that a highly accurate assessment of poly(A) tail length can be obtained from a wide range (higher or lower) of input mRNA amounts. Care should be taken to avoid signal saturation on the gel imaging system thus making an accurate determination of poly(A) tail length more difficult.
However, it is recommended to normalize input mRNA amounts due to the ability of the assay to determine not only the length of the poly(A) tail, but also the intensity of poly(A) tail length distribution regions which can serve an informative and diagnostic purpose to determine reaction efficiency and optimization.
A: The Poly(A) 20-mer Ladder will extend to extremely high poly(A) lengths of >500-nt. The Poly(A) 20-mer Ladder contains a 100-nt DNA oligo marker for orientation of the band sizes. When assaying very long poly(A) tails, it is likely that the gel will need to be run for an extended amount of time which will run the 100-nt DNA oligo marker off of the gel. If you anticipate such a situation, we recommend running an additional RNA molecular weight marker (e.g., RNA Century™ Marker, Thermo Scientific) on the gel to orientate to the proper size of the Poly(A) 20-mer Ladder bands. Note: mixed NTP RNA ladders will not comigrate precisely with the same length poly(A) ladder bands with the differences in mobility increasing as the band length increases. The 100-nt DNA oligo marker migrates slightly faster than the 100-nt poly(A) band, while a mixed NTP RNA marker band will migrate slightly slower than the equivalent length poly(A) marker band.
A: RNase A will digest all RNA given enough time and/or high enough concentration. However, it preferentially cleaves after pyrimidines, and as such has been shown to not be damaging to the standard 5 pmol of input mRNA in the presence of vast excess (up to 10-fold) of RNase A for 2 hours. The likelihood of deteriorating poly(A) tails in an extra hour of digestion at the recommended enzyme concentration is extremely low.
A: If the Poly(A) 20-mer Ladder bands are dim, ensure that 5 µl ladder are being loaded onto the gel. Additionally, ensure the Poly(A) Fluorescence Enhancer is being used in the staining process.
If only the sample bands are dim, then several possibilities might exist.
- Insufficient tailing: Enzymatic tailing can be reperformed on the mRNA and then the assay repeated. If template-encoded tails were used, evaluation of full-length product may help to rule out problems with the in vitro transcription step.
- Insufficient sample was loaded: Ensure that the correct amount of input mRNA was used in the RNase A digestion step.
A: Bowing or unevenness of gel bands is indicative of insufficient washing of the wells of the gel prior to loading due to urea settling in the gel wells. Clean the gel wells by flushing with gel running buffer using a pipette immediately before loading your samples.
A: The presence of multiple bands within a single lane, particularly a notably smaller band, typically indicates the production of short RNA transcripts during the in vitro transcription (IVT) reaction. These small transcripts, (which range in size from ~3-25-nt) may be sense (i.e., abortive transcripts) or antisense (generated by RNA-dependent, promoter-independent mechanisms during transcription). They become polyadenylated during the poly(A)-tailing reaction of the mRNA synthesis process. The resultant tails on these fragments are shorter than the poly(A) tails generated on the primary transcript and thus, once released during the RNase A digestion of the mRNA sample, also appear on the poly(A) tail length determination gel. Disregard the band(s) when determining the length of the poly(A) tail on the mRNA sample being tested. Note: confirmation of this process can be shown by running a sample of non-RNase A digested mRNA on the gel alongside the RNase A digested mRNA sample. The shorter band should appear at approximately the same position in both lanes, as it consists primarily of a poly(A) tail. For a visual example, please refer to the troubleshooting gel images provided in the kit documentation.
A: Poly(A) tails are simple nucleic acid strands and so can be run on any other instrument with specialized methodology or equipment to handle them with certain caveats.
- HPLC-MS has size restrictions and buffer restrictions that must be met. If the poly(A) tail is too long, this run may not be feasible. The Stop/Loading Buffer that is added to the sample following RNase A digestion contains formamide and may not be compatible with some instrumentation. Saving a direct aliquot of the completed reaction sample prior to adding Stop/Loading Buffer followed by storage at -20oC or -80oC may be necessary to halt RNase A activity without adding formamide.
- Due to the resistance of poly(A) tails to uptake dyes used in Fragment analyzers or Bioanalyzer, care should be taken when loading them onto other platforms.
