As a methods-oriented journal, we are particularly aware of, and often primarily focused on, the details that authors provide in the Materials and Methods section. While it should be very obvious why we do this (nothing is worse than being unable to replicate a method described in the pages of a “methods journal”), I for one think that authors should pay close attention to how they describe the materials and methods used for any study, regardless of the journal for which it’s intended.
The Materials and Methods section should not simply be an afterthought or, as many authors seem to think, a nice way to ease into writing the rest of the manuscript. Unfortunately, the rise in the complexity and scope of Materials and Methods sections, along with the need to reduce the size of articles appearing in print, has led to a trend of pushing almost (if not) all of the Materials and Methods into the online Supplementary Materials. As a result, authors assign less importance, and less time, to writing this part of the manuscript.
I hope by the end of this column you will agree that neglecting the Materials and Methods section could be a disservice to both authors and readers in the long term. And since the trend of online Supplementary Materials is not likely to change anytime soon, I thought I should devote this column to some basic but essential tips for putting the most important details into a Materials and Methods section.
When you start to write up your Materials and Methods, the question that you need to ask is: How much information would someone else require to repeat the experiments done in my manuscript? And remember, when you answer this question, you cannot cheat by assuming that the reader knows everything you do—you need to ensure that all of the necessary information is included and that it is put in the proper order.
Let’s begin by examining a Materials and Methods section from a recent article. Do you think there is too much information, too little, or just the perfect amount in this case?
"Individual E. coli colonies were inoculated in LB broth, incubated at 37°C with shaking for 3 h, and centrifuged at 4°C. RNA was extracted from the cells using a standard kit. 1 µg of E. coli total RNA was treated to deplete rRNA sequences. Intracellular RNA was extracted from human PBMCs (Astarte Biologics, Redmond, WA, USA) using RNAzol followed by cleanup and elution into nuclease-free water. Total RNA extracts were visualized and RNA integrity values were determined. Triplicate samples containing approximately 300 ng of E. coli K-12, untreated and Ribo-Zero treated, and human PBMC RNA were fragmented by incubating with a magnesium chloride buffer to obtain a fragment size range of 25–300 bp. RNA fragments were purified and eluted in 6 µL of nuclease-free water."
If we break the paragraph down, it is easy to see that the basic information is there: the bacterial strains, the basic culture conditions, and the methods for extracting and quantifying the RNA. Now, we pose the important question of whether or not this is enough for someone else to repeat the experiments exactly as the authors did (or as close as possible), since in the end it is this level of detail that is needed for a Materials and Methods section.
Looking at the above paragraph, a couple of questions quickly come to mind. “RNA was extracted with a standard kit” prompts the question of what kit was actually used in the study; “RNA was treated to deplete rRNA sequences” obviously leads one to wonder how this depletion treatment was done. If you read the paragraph again, I hope it will quickly become clear that there are multiple sentences where the details are vague or sketchy, which could cause confusion for novice, or even experienced, researchers hoping to replicate the study.
So, how might more detail and explanation enhance the impact of this paragraph?
"E. coli strain K-12 was obtained in lyophilized form from ATCC (Manassas, VA, USA). Bacteria were cultured with addition of 300 µl of LB broth (BD, Franklin Lakes, NJ, USA) followed by plating on LB agar (BD) and incubation at 37°C overnight. Individual colonies were used to inoculate in LB broth, incubated at 37°C with shaking for 3 h, and centrifuged at 5000× g (RCF) at 4°C for 5 min. RNA was extracted from the cells using the RNeasy Protect Bacterial Mini Kit with on-column DNase treatment (Qiagen, Valencia, CA, USA). 1 µg of E. coli total RNA was subjected to Ribo-Zero treatment to deplete rRNA sequences using the Gram-Negative Bacteria kit following manufacturer's instructions (Epicentre, Madison, WI, USA). Total intracellular RNA was extracted from human PBMCs (Astarte Biologics, Redmond, WA, USA) using RNAzol (MRC, Cincinnati, OH, USA) followed by Directzol column cleanup kit (Zymo, Irvine, CA, USA) and eluted into nuclease-free water. Total RNA extracts were visualized and RNA integrity values were determined using a 2100 Bioanalyzer RNA nanochip (Agilent, Santa Clara, CA, USA). Triplicate samples containing approximately 300 ng of E. coli K-12 (RIN >9.0), untreated and Ribo-Zero treated, and human PBMC RNA (RIN >8.5) were fragmented by incubating with a magnesium chloride buffer (New England Biolabs, Ipswich, MA, USA) for 5 min at 95°C to obtain a fragment size range of 25–300 bp. The RNA fragments were purified using an RNA clean and concentrator kit (Zymo), and eluted in 6 µL of nuclease-free water."
This paragraph is obviously longer and more detailed than the first. While increasing the word count might seem to be moving away from the concise structure we have suggested in our previous articles on the Abstract and Introduction sections, I would argue that, when it comes to the Materials and Methods, take the space you need to be as clear as possible (plus, many journals do not place word count limits on Supplementary Materials).
The second version of the paragraph answers those questions raised when we looked more closely at original version. Now we have details on the RNA extraction methods used, as well as how rRNAs were depleted from the RNA extract. We know the exact kits used (something that I think goes far in helping replicate methods but, depending on journal, may or may not be required) and the manufacturers of those kits, as well as exact volumes, times, and temperatures for specific steps in the protocol. This paragraph works because, although longer, it is actually a very concise, accurate description of the way in which the study was conducted.
A final thought: It can often be easy to gloss over the Materials and Methods and assume your readers are familiar with certain techniques or approaches. This way of thinking is not only unfair to the reader; it could also hurt you as an author and a researcher when others fail to replicate the results due to less than thorough descriptions of your methods.
When it comes to Materials and Methods, more is always better—especially in the era of Supplementary Materials. In our next manuscript writing tips column, we will explore how to discuss your data.
BioTechniques Special Series: Manuscript Tips