Intro to human blood components, cell types, and cell markers

Blood components as separated by density gradient

1. Plasma: yellow layer on top, 55% of blood volume

2. Buffy coat (leukocytes and platelets): white layer in the middle, <1% of blood volume

• White blood cells (leukocytes) = granulocytes (neutrophils, eosinophils, basophils) and PBMCs/agranulocytes (monocytes, dentritic cells, T cells, B cells, NK cells)
• Platelets (thrombocytes) = the blood clotting cells, the smallest blood cell type
• Related: buffy cone refers to the leukocyte-rich waste product from plateletpheresis procedures. It isn't the same as buffy coat , which is a layer from the density gradient, but both are sources of leukocytes.

3. Red blood cells (erythrocytes): red layer at the bottom, 44-45% of blood volume. 

• Adult red blood cells are anucleated (don't have a nucleus). The nucleus is lost during cell maturation.
• Fetal red blood cells do have a nucleus . This affects cell density makes it more difficult to use density gradient medium protocols to separate cord blood into the three layers, compared to adult peripheral blood.

Learn more about blood components and blood cell types:

• Blood (human), MACS Handbook by Miltenyi Biotec. Includes useful figures and tables.
• "What is buffy coat in blood? Buffy coat preparation and buffy coat cell extraction" by Akadeum Life Science
  

Buffy coat components (leukocytes and platelets)

Vocabulary for reading DNA methylation papers

 If you are new to the field of DNA methylation, here is some introductory vocabulary.

• 27k array - short for Infinium HumanMethylation27 BeadChip (approx 27,000 methylation sites). An early DNA methylation arrays that samples CpG sites across the human genome. Not used anymore. 
• 450k array - short for Illumina HumanMethylation450K BeadChip (>450,000 methylation sites). This used to be the gold standard for array-based DNA methylation measurements for several years, though now the EPIC array is available with more methylation sites.
• Beta value and M value are two terms used to describe the methylation measurement.
• Beta values range from 0 to 1 and describe the proportion of methylation for a specific site in the sample, from completely unmethylated to completely methylated. 
• M values can be negative or positive and are a result of data normalization. M values are more useful than beta values as input for statistical models. If you want to describe differentially methylated probes (DMPs) then you need M values for your analysis.
• Beta values or sometimes "delta beta", annoyingly, can also refer to the model coefficient result from a generalized linear model. In that case, they would describe if a site is more or less methylated in group1 versus group2. Positive and negative values refer to the direction of higher methylation. Consult the manuscript to figure out the direction.
• Bisulfite conversion - this is a chemical change done by the lab to make it easier to distinguish unmethylated versus methylated sites on the DNA. It is done before the DNA methylation measurement step, whether that be bisulfite sequencing or bisulfite PCR or a methylation array. Bisulfite conversion does NOT mean bisulfite sequencing.
• Bisulfite sequencing - this is a method of measuring DNA methylation in a sample, using bisulfite conversion followed by DNA sequencing. 
• Bonferroni correction - this is a way of correcting p-values when you have a lot of measurements and thus a higher risk of false positives. The Bonferroni method is much stricter than the FDR method.
• P<0.05 is "nominally" significant, meaning it seems significant for a single site or gene but if you're looking at many sites or genes then you have a higher chance that your data has some false positives, and therefore P<0.05 isn't good enough for big data. You need to adjust for multiple comparisons to cut down the risk of false positives.
• FDR<0.05 is actually significant for big data.
• Bonferroni<0.05 is actually significant for big data and much stricter than FDR<0.05
• Bonferroni<0.05 data points are also going to meet criteria for FDR<0.05 and P<0.05, since Bonferroni<0.05 is stricter than both
• CpG - dinucleotide grouping of a cytosine base followed by a guanine base on the same DNA strand. It is not the same as C and G binding across DNA strands. CpG methylation (with a methyl group on the C base) is what people usually mean when they say DNA methylation, since it's the most common form of DNA methylation.

Study questions for sequencing research articles

Study questions for a sequencing article:

What is the article citation? Include the full article title, a PubMed link to the article, and a link to the article's original publisher.

Who is the corresponding author and what is their institutional affiliation? The corresponding author is usually the last author on the author list. They will have an asterisk or another symbol by their name. This is the person who oversees the project and makes publication decisions.

What samples did the study use (tissue type and gestational age and source of sample)? Define any acronyms used. Some examples:

• Placenta - what gestational age? If pre-delivery, were the samples from terminations or from continuing pregnancies? 
• Cord blood - this is collected at delivery. Were the pregnancies "full term" or "pre-term" or otherwise described?
• Plasma - when collected?
• Serum - when collected?

Did they collect the samples themselves? If they are analyzing data from another paper, what is the citation for that other study? Check methods to see if they indicate an NCBI GEO accession ID (begins with "GSE").

What sequencing or array method(s) did they use? Some examples:

• total RNA-sequencing: sequences all RNAs after depletion of ribosomal RNAs.
• mRNA-sequencing: only sequences the polyadenylated RNAs (with polyA tails) using oligo(dT) primers or probes during the library preparation.
• miRNA-sequencing, microRNA-sequencing, or small RNA-sequencing: sequences small RNAs within a specific size range.
• ATAC-sequencing: sequencing to identify accessible chromatin regions, where DNA is available for protein binding. This is used to find regulatory DNA regions and identify how genes might be controlled.
• Bisulfite sequencing: a sequencing-based method to measure DNA methylation
• DNA methylation array: an array-based method to measure DNA methylation, but which one? The two most common arrays are the 450k array (older) and the EPIC array (twice the size at 860k sites). Some papers also use custom arrays.
• Vocabulary to help distinguish methods:
• "Total RNA" = all RNA isolated from the sample before any depletion or enrichment step. The use of the term "total RNA" does not indicate the sequencing method. Most RNA in cells is actually ribosomal RNA so pre-sequencing steps usually include a negative selection (remove rRNA) or a positive selection (pick up only polyA-tail RNA).
• Library preparation is the step to convert RNA into cDNA before RNA-sequencing. The cDNA "library" is what is actually sequenced.
• DNA is bisulfite converted for methylation arrays also, so the word "bisulfite" in methods does not by itself indicate bisulfite sequencing.

Cell biomarkers in placenta research

Context is important for cell markers. Before you use these markers, consider that KRT7 is a marker for trophoblast cells in placenta, but you would not say that any particular cell is a trophoblast just because it expresses KRT7. It is also a common marker for lung research even though lungs don't have any trophoblasts. That is because KRT7 is generally expressed by epithelial cells, and in placenta those are trophoblasts, but lungs have other epithelial cells. Sample context is important.

Also consider RNA vs protein, localization, and experiment use. Some markers are useful to identify cell types in single cell RNA-seq results because the RNA is specifically or mostly expressed by specific cell types, but the protein is not expressed at the cell surface so you wouldn't use that marker to sort cells for flow cytometry. 

• KRT7 = CK7 = Cytokeratin 7 = epithelial cells, trophoblast cells
• Expressed by all epithelial cells
• All trophoblast cells in placenta are epithelial, so KRT7 is used as a trophoblast cell marker in placenta tissue
• qRT-PCR marker
• Cell surface marker suitable for flow cytometry
• Beware that fibroblasts also express KRT7 and primary cells in culture will eventually become fibroblast cells

• KRT8  = epithelial cell marker, trophoblast marker
• All trophoblast cells in placenta are epithelial, so KRT8 is used as a trophoblast cell marker in placenta tissue

• CDX2 = marker for early cytotrophoblasts (CTBs)
• Expressed in CTBs around 6 weeks, but diminishes as 1st trimester progresses (Horii et al 2016).
• Indicates stem cell-like phenotypes
• Immunohistochemistry (IHC) and gene expression marker for early cytotrophoblasts

Placenta research 101

Quick primer for new lab members. Placenta genetics, cell lines, and vocabulary you'll hear in lab meetings.

Nomenclature of microRNAs

Mature microRNAs

The full name of mature microRNAs is usually written like this (no bold needed):

hsa-miR-202-3p

• hsa = Homo sapien species abbreviation
• Warning! Microsoft autocorrects "hsa" to "has"
• If you see the "has" prefix in a publication, it's likely a typo
• Add "hsa" to your spellcheck dictionary
• miR = abbreviation for microRNA used when naming the RNA sequence
• MIR, all capitals, is used for gene names only, not the RNA transcript
• Not to be confused with "piR", which refers to a different class of RNAs called PIWI-binding RNAs or "piRNAs". These do not bind the same proteins as miRNAs.
• 202 = code for the precursor microRNA which can be processed into two mature microRNAs (3p or 5p)
• 3p = indicates that the mature miRNA (21-24 nt) comes from the 3-prime end of the hairpin structure