A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach
Round 1
Reviewer 1 Report
In Ahmad et al. manuscript, a comparison of conventional northern blot versus liquid hybridization was made. The authors showed that liquid hybridization is a more sensitive, efficient, cheaper, and faster method for detecting large and small RNA molecules. The manuscript describes the methods and the obtained results in detail and certain modifications of the methods to make the obtained results more reliable.
Some additional explanations and corrections should be made before publication.
- In the Introduction section - Figure 1 is shown that EDC is/was used in liquid hybridization, while in the Material and methods section, line 817-818 is written that EDS is used in conventional Northern blot. The authors should explain this discrepancy. Moreover, in Discussion, line 547, authors stated that EDC is for conventional Northern blot, while in lines 563-564 is written that is used in liquid hybridization. Why did the authors recommend ECD only for conventional Northern blot when it is stated that ECD had a similar effect on liquid hybridization samples?
- Line 309 - word 'gels' is written twice
- Line 524 - word 'been' is written twice.
- Figure 10a - are those actual results, or are they a bit modified? The bands are too perfect to be true.
- Figure 11h is not described in the figure legend.
- Line 521 - typo porbe - probe
- Lines 746-748, the same sentence is written twice and should be deleted.
Author Response
Please see the file uploaded for a point-by-point response.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This is an interesting technical paper presenting the merits of liquid hybridization (LH) against northern blot (NB) method for the detection of various sizes of RNA molecules. There is a lot work done by the authors both at wet lab and manuscript preparation, however the current status of the manuscript does not reflect the quality of published material in CIMB.
My suggestions and concerns are listed below:
The manuscript and the figures are prepared by more than one people and there is evident variation in the use of English language and format. Team work is encouraged but one person must coordinate and polish the manuscript when completed. I suggest careful proof reading so as the text is as neatly written as the introduction. Apply the same format on all figures. Change ug to μg, nts to nt.
It is not really conventional northern if radioactive probes are not used. I would change Conventional northern to Northern blotting. There is no reason to present this work as a battle between NB and LH. As authors state both methods have merits and limitations. Expressions as “cumbersome”, “fallen back” could me omitted.
It is useful to report in the text what tissue or cell type the RNA is extracted from, not just the figure legend.
In order to compare the sensitivity potential of the two methods, the membrane exposure times should be mentioned. Care must be taken when obtaining signal with autoradiography film, signal can saturate at certain over concentrated samples and afterwards the measurements will not present a linear range. Experiments have to be repeated 3 times at least and be statistically analyzed.
On figure 4(b): the β-actin film should be flipped vertically, the result on c-Myc film cannot be apprehended, presented or analyzed as such.
The graph on figure 7(a) does not reflect the experiment presented in 7(b). A B C D stands for numbers mentioned on the legend that are not used in LH detection of 5S RNA.
microRNA precursors are highly structured in hairpin-like conformations that might escape access from LH probes. The LH protocol should be optimized for such RNAs. Denaturing PAGE used in NB allows for pre-miRNAs to be detected. One cannot assume that the lack of signal for a miRNA precursor is direct evidence that this miR matures quickly (Line 431). A positive control is needed.
The methods section is well written, however there is excess technical information through the results section and the figure legends that is repetitive and unnecessary.
I would reconsider the acceptance of this work to CIMB provided that the points described above are revised.
Author Response
Please see the file uploaded for a point-by-point response.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I am very pleased with the effort made to improve this manuscript. My points were adequately answered and I am ready to suggest this work for publication in CIMB.
