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Both Reviewers found that their concerns were properly addressed and the method described in the revised manuscript is very useful for the tendon field.
# PeerJ Staff Note - this decision was reviewed and approved by a PeerJ Section Editor covering this Section #
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Thank you for the edited manuscript. You addressed all of my previous concerns. This method will be very useful for the tendon field.
This is a well written study with appropriate references and objectives.
The experimental design is appropriate and the techniques are well described.
The findings are valid, and the data is robust and statistically sound.
This paper will be a nice addition to the tendon literature.
Your manuscript was reviewed by two Reviewers. Both Reviewers found your cost-effective RNA isolation method from a single adult mouse tendon beneficial for the scientific field. From my own reading of the manuscriptf, I agree with the reviewers that this is a carefully designed and executed work and a well-written manuscript, which describes a simple, cost-effective, and efficient method for obtaining high-yield and high-quality RNA from a single mouse tendon.
Please revise your manuscript based on the Reviewers' comments.
Please also send your point-by-point responses to the comments of Reviewer 1. Considering these comments will increase the quality of your paper.
As PeerJ does not offer copyediting, I also suggest to check carefully your manuscript for typing errors.
- Typo on line 237: keep use of "p" vs "P" consistent
- Typo on line 275: "repeats" should be "replicates", correct?
- The authors did a great job in establishing previous research in the field regarding extracting RNA from tendon tissue. While RNA amplification (line 86) can be quite costly, especially for RNA Seq applications, there are more cost-effective methods of pre-amplification for qPCR. One such method is Specific Target Amplification (STA) offered by Fluidigm: https://www.fluidigm.com/search?query=specific%20target%20amplification. (Example reference: Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., et al. (2011). Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. BMC Genomics, 12(1), 144). It would improve the article to refer to this technique.
- The authors presented results from other disruption methods (lines 187-199) including "enzymatic digestion, cryogenic grinding (manual and mill), shearing with a handheld homogenizer". Would you please provide additional details in the methods or supplemental methods regarding these procedures? For instance, which enzyme was used, concentration, time, etc.?
- When the sample was placed on ice prior to homogenization (line 203), was it in a solution such as trizol? Or was it in air? Please add this detail to manuscript.
- The authors state that homogenization times longer than 360 seconds could cause RNA degradation (lines 215-216). How much longer was required to degrade the RNA? This would be useful information to include.
- The authors stated in the methods (line 161) that nanodrop readings were taken. What were the 260/280 ratios for these samples? Was the RNA sufficiently pure with minimal contamination that may hinder RNA Seq library prep?
- I would suggest changing title for Fig. 3 to "Tendon pooling affects RNA yield but not quality" which better matches what is stated in the results section.
- While I agree that RNA Seq experiments can be conducted for RNA samples with RINs ~ 6.5, the authors should mention that this level of quality may not be sufficient for certain library preps. For instance, a RIN of 6.5 is borderline in terms of being sufficient for mRNA sequencing where PolyA selection occurs. Most manufacturers recommend RINs > 7 for this type of sequencing (e.g., Illuminia TruSeq RIN > 8, SMART-Seq RIN > 8, Exiqon RIN > 7). However, 6.5 is more than sufficient for total transcriptome sequencing using random priming libraries.
The authors present an inexpensive, simple method for extracting RNA with high yield and high quality from single murine tendons. As the authors stated, pooling of samples is commonly performed to obtain sufficient levels of RNA per biologic replicate. Being able to efficiently isolate enough RNA for downstream analyses without compromising quality or requiring expensive pre-amplification methods, would benefit the field.
This is a well-written manuscript. The topic of improving RNA yield from connective tissue is important in the musculoskeletal field.
The experimental design is excellent and the methods are well described. Although not possible to test every permutation, and some experiments were limited in sample size, this paper provides useful observations as a technical note.
The findings are useful in demonstrating their RNA isolation approach provides improvements over many of the currently utilized techniques.
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