Month: October 2018

As discussed in a recent blog post, RNAi is a common technique used in the Applewhite lab to observe the effects of a silenced gene. When preparing for RNAi, it is common practice to run a sample of the dsRNA template on a gel to make sure the resulting band is the same size as the target. However, is this sufficient practice to conclude your following results are due to the knockdown of the gene? Many publishers would say no. It is possible that the exogenous dsRNA was not in appropriate concentration, or was not an effective target to cleave the specific mRNA sequences. Real time quantitative polymerase chain reaction (qRT-PCR) is a supplementary method used to verify the successful knockdown of the gene of interest.

Quantitative PCR (qPCR) is accomplished by extracting endogenous RNA from your cells treated with RNAi, reverse transcribing the RNA to DNA, designing primers to amplify the gene of interest, and using intercalating dyes, such as SYBR Green, which bind to the DNA and fluoresce with greater intensity as the concentration of the target sequence increases. Important to note, in the presence of off-target dsDNA, sequence-specific probes can be used which rely on FRET for detection, and fluoresce only when the DNA polymerase separates the quencher from the emitter. These sequence-specific probes include Taqman, Molecular Beacons, and Scorpions, although require more complex and expensive implementations (1).

The real time element is essential to determining initial DNA template concentration. Since qPCR only measures the end concentration of target sequence, there is no way to calculate an initial concentration. qRT-PCR however, measures template concentration at an exponential stage of replication, which allows for calculation of an initial starting concentration. This in turn, enables analysis of initial gene expression, and if minimal, verification of RNAi success (2).

Figure 1. Relative qRT-PCR and qPCR measurements of target concentration in respect to duration of PCR.

Sources Cited

1. The Basics: RT-PCR.Thermo Fisher Scientific – USAvailable at: https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rtpcr-analysis/general-articles/rt–pcr-the-basics.html. (Accessed: 23rd October 2018)
2. Bansal, R.et al.Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug.Plos One11,(2016).

 

One aspect of my thesis is exploring co-localization of Split Discs with other proteins in drosophila cells. In order to do this, not only does wet lab work need to be accomplished, but mathematical analysis (in this case using Mander’s coefficient).

Fluorescence microscopy does not have the ability to see whether or not two molecules are directly interacting. However, by looking to see if they co-localize in the cell, it can be determined whether they interact with the same complexes in the cell. The limit for fluorescence microscopy is the resolution of the images produced. Because of this, small numbers of puncta are not sufficient for determining whether or not the experimental molecules are actually co-localized. Multiple puncta from different regions within the cell must be used in analysis so the data is not limited to overlapping puncta which are a result of organelles that are close in proximity to one another.

In order to quantitatively determine the correlation of co-localization in the cell, mathematical analysis of the data is employed. For my thesis, I am employing Mander’s Overlap Coefficient (MOC) for this analysis because it does not require distinguishing fluorescence as being the result of a fluorescent protein or background noise. MOC is able to do this because it only compares the co-occurrence of fluorescence among pixels. MOC = ∑i(Ri×Gi) / √(∑iR2i×∑iG2i) where Ri and Gi are the average level of grey from the red and green fluorescence respectively (Manders et al., 1993). MOC has a range of 0 – 1 and Ri and Gi have a range of -1 – +1. The limitation to this equation is that the ratio of values can result in ambiguous numbers. Therefore, the numerator and denominator can be split up in such a way to account for the ambiguity.  From this we get two coefficients: M1 (fraction of red fluorescence in areas with green fluorescence) and M2 (fraction of green fluorescence in areas with red fluorescence)  (Manders et al., 1993). M1 = (∑iRi,colocal) / ∑iRi where Ri,colocal = Ri if Gi > 0 and Ri,colocal = 0 if Gi = 0 and M2 = (∑iGi,colocal) / ∑iGi where Gi,colocal = Gi if Ri > 0 and Gi,colocal = 0 if Ri = 0 (Manders et al., 1993). The larger MOC, M1, and M2 are the stronger the evidence for co-localization of the proteins within a cell. In my thesis, MOC, M1, and M2, will be gathered for each cell to determine whether or not Split Discs are co-localizing with other specific proteins.

 

References:

 

Dunn, K. W., Kamocka, M. M., & McDonald, J. H. (2011). A practical guide to evaluating colocalization in biological microscopy. American Journal of Physiology – Cell Physiology, 300(4), C723–C742. http://doi.org/10.1152/ajpcell.00462.2010

Manders, E. M., Verbeek, F. J. & Aten, J. A. (1993). Measurement of co‐localization of objects in dual‐colour confocal images. Journal of Microscopy, 169, 375-382. doi:10.1111/j.1365-2818.1993.tb03313.x