An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women

Round 1

Reviewer 1 Report

This is a very interesting article regarding mRNA profiling of vaginal swabs from pre and postmenopausal women. The article is in general well written, and of interest for forensic practitioners. 

There are some limitations regarding this method, at least for practical purposes, which should be clearly emphasised in a special subchapter, regarding limit of the study.

 

The English is fine

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The present study aims to expand the analysis published by the same group (ref. 17), integrating all the results with those subsequently received from a series of 10 laboratories integrating the so called “International Society for Forensic Genetics (Genetisti Forensi Italiani, GeFI). The study focused on a set of proposed forensic vaginal mRNA markers (CYP2B7P1, CytochromeP450, family 2, subfamily B, polypeptide 7 pseudogene 1, MUC4, mucin 4 and MYOZ1, myozenin-1) as part of the 19-plex mRNA profiling assay previously established. Under my opinion, a brief description of the choice of these 3 genes would be convenient. Anyway, the aim is worthwhile, and methods, interpretation and statistical analysis seem to be correct, although the details of the ten laboratories are not given.

As a general comment, this is a descriptive rather than interpretative report. Some labs seem to give quite different results, so that it would be very important that all labs offer similar profile. Some of the percentages described point out the results are provisional, and they need improvement improved yet. On the other hand, final conclusion is correct, although not totally determinant. It is confirmed that mRNA profiling can be a precious molecular tool for the reconstruction of crime dynamics, especially in sexual assault cases. However, routine implementation of forensic mRNA assays remains a work in progress and further validation is needed to refine analysis.

 

Minor points to be addressed before acceptance:

1.    Concerning the following two statements:

(i)    Referred to FIG 2. Lines 180-182 a significant negative correlation (p = 0,049, Spearman rank test) was exclusively observed for marker MUC4 in the PR-M subset. And soon later:

(ii)   Referred to FIG 3: Lines 197-198: A significant increase in average relative contribution to total peak height was observed for MUC4 in PO-M samples, compared to PR-M samples. 

In fact, both statements are the same, but I think this can be related to Figure 3, but not to Figure 2. Thus, I suggest deletion of lines 180-182, that are premature, and leave lines 197-198.

On Table 1: The line numbering (225---) have been shifted to the Table. Please, repair the format. The meaning of PR and PO at Table S1 pre- (PR-M) and postmenopausal (PO-M) should be reminded for facilitating comprehension. As suggestions: a) Please, clarify the meaning of not observed at the legend. Alternatively, as the sum of “observed” and “not observed” is obviously 100%, the first column of the Table would be enough. This point is also applied to Table 2:

On Table S2: the units for optimal cDNA input should be given. Microliters taken from dried swabs are ambiguous confusing terms unless more details would be given. It seems that 9 labs use one protocol, but the 1 left uses a different one. If possible, please, clarify in terms of ng of DNA.

Lines 252-254: It is suggested that some RNA degradation was likely. Thus, the time of storage could be important...and the samples are kept 6 to 20 months at room temperature. However, at line 286, at the beginning of discussion. (line 287), it is stated the prolongued stability of mRNA in vaginal samples. This point should be discussed. properly

 Finally, please, some abbreviations, such as CDSN, should be defined.

English shoud be edited. Some sentences would be re-written

Author Response

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Author Response File: Author Response.pdf