Designing Primers for Alternative Transcripts #1

Genes are usually expressed as multiple alternative transcripts as a result of alternative splicing of the pre-mRNA. Pan primers amplify all transcripts so the forward and reverse primers are designed to anneal to exons that are common to all transcripts (see figure below).

In order to amplify different alternative transcripts via PCR or real-time PCR, you may need to custom design your own primers if what you need are not commercially available.

Assuming that you are working with a known gene sequence and that you also know which alternative transcripts you want to amplify, you will need to design at least one of your primers, either forward or reverse, to span across adjacent exons. 

For example:

The forward primer spans across exons 1 and 3. This makes the forward primer specific for transcripts which lack exon 2.

To design primers, refer to Primer Design.

Ways To Ensure Experimental Consistency #3

Commercial Kits

There are many commercial kits available to streamline and simplify various experimental procedures including DNA/RNA extraction, plasmid preparation, cloning, immunoassays, reporter assays, and so forth.

Commercially available kits are certified for quality and have proven efficacy so long as the instructions for use and handling are adhered to. However, there are a few important points to note in regards to reagent and sample preparation, points that some product manuals may not necessarily provide information on. 

* Ensure that components of buffers and reagents are in solution. With some buffers, you may see precipitates. For example, lysis buffers containing SDS tend to develop white precipitates when the room temperature is low. This is the SDS falling out of solution. In such instances, warm up the buffer in a water bath before use. 

* Ensure correct storage of kit components. Make sure that after opening and using individual kit components that they are stored at the recommended temperature. While a kit may arrive at room temperature, after a buffer is opened it may need to be stored at 4 degrees.

* Note the expiry date of the kit and/or specific components.

* If you are required to prepare a component that is not provided (e.g. addition of 3’ A overhangs to inserts for TOPO cloning) be sure to use reagents that are of a quality and purity compatible with subsequent parts of a kit. 

* Ensure that samples are extracted in a buffer that is compatible with the reagents supplied. A notable example is the Bradford Protein Assay, which is incompatible with some detergents and substances.

Before Cloning….

For using restriction enzymes to digest and ligate genes of interest into plasmid vectors, it is good practice to check if your restriction enzyme(s) of choice also cuts at regions other than those intended. For standard commercial vectors, these usually have a multiple cloning site (MCS) region containing unique restriction sites where the corresponding enzyme will only cut. However, if you opt to use an enzyme whose site is not present in the MCS, you will need to check if your enzyme(s) of choice cuts anywhere else on the vector.

For your insert, if you decide to engineer restriction sites onto the 5’ and 3’ ends, it is better to (1) choose sites that do not cut anywhere in the insert and (2) make sure the restriction site you are incorporating correspond to what is present in the MCS of the vector.

Some good online tools that are freely available to use for identifying restriction sites in DNA sequences include:

* NEBcutter v2.0

* RestrictionMapper v3

* Webcutter 2.0 

Calculating the Geometric Mean/Geomean of Housekeeping Genes from Real-Time PCR Data Using Excel

In real-time PCR, it is not uncommon for multiple housekeeping genes to be used for normalising data. Knowing how to calculate an average for your housekeeping genes will be useful regardless of whether you opt to carry out relative or absolute quantification of your gene of interest.

If you are using three or more housekeeping genes, you can calculate the geometric or geomean easily using Excel.

Cell Formulae

Column B: Gene 1 CT values from your qPCR run

Column C: Gene 2 CT values from your qPCR run

Column D: Housekeeping gene 1 CT values from your qPCR run

Column E: Housekeeping gene 2 CT values from your qPCR run

Column F: Housekeeping gene 3 CT values from your qPCR run

Column G: =GEOMEAN(D4:F4) and copy/paste formula down to G18

Epigenetics – The nth DNA Base

Classically, DNA is thought to comprise 4 nucleotide bases: adenine, thymine, cytosine, and guanine (A, T, C, and G). However, the discovery and identification of variants of the classical 4 bases including 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in human and mouse brain tissues has revolutionized the conception of DNA, its composition, regulation and expression.

While the sequence of the 4 classical bases determine what genes encode, the additional bases are involved in controlling how DNA sequences are interpreted, what genes are expressed, and importantly when genes are expressed. For instance, epigenetic modifications made to cytosine can affect the DNA structure by exposing regions of the DNA to attract different proteins and transcription factors, which could either directly or indirectly influence which genes are expressed or repressed.

* 5-Methylcytosine is formed by the addition of a methyl group to the 5th carbon of cytosine. This process typically occurs at cytosines located in CpG dinucleotide sequences, although, methylation has also been found to occur at non-CpG dinucleotide sites. 5-Methylcytosine has been shown to function as a repressor of gene transcription. In the promoters of genes, 5-methylcytosine is associated with stable, long-term transcriptional silencing. By enabling genes to be turned on and off in specific cell types, the gene regulatory function of 5-methylcytosine is an important mechanism in mediating genomic imprinting, controlling cellular differentiation, and the expression of specific genes for normal tissue patterning and development.

* 5-Hydroxymethylcytosine is abundant in the brain and in embryonic stem cells. It is formed by oxidation of 5-methylcytosine and reduced levels of this cytosine variant in DNA has been regarded as a hallmark of cancer. Genomic profiling of 5-hydroxymethylcytosine has revealed that in contrast to 5-methylcytosine, 5-hydroxymethylcytosine is particularly associated with gene regulatory elements where 5-methylcytosine is depleted. 5-Hydroxymethylcytosine has been found to be associated with cell proliferation, having been demonstrated to form immediately during DNA replication at the stage of synthesis via isotopic labelling studies of DNA in mouse tissues.

* 5-Formylcytosine is derived from 5-methylcytosine by Tet-mediated oxidation. In mice, 5-formylcytosine has been found to be present in all tissues, including embryonic tissues, and preferentially occurs at poised enhancers among other gene regulatory elements. 5-Formylcytosine has been implicated in various roles including demethylation, chromatin remodeling, and DNA structural changes such as changes to groove geometry and base pairs associated with 5-formylcytosine-modified bases that lead to helical underwinding.

References:

1. Cadet and Wagner. (2014) TET Enzymatic Oxidationof 5-Methylcytosine, 5-Hydroxymethylcytosine and 5-Formylcytosine. MutationResearch/Genetic Toxicology and Environmental Mutagenesis 764–765: 18–35.

2. Wanger et al. (2015) Age-DependentLevels of 5-Methyl-, 5-Hydroxymethyl-, and 5-Formylcytosine in Human and MouseBrain Tissues. Angewandte Chemie (International Ed. in English) 54:12511–12514.

3. Yu et al. (2013) DevelopmentallyProgrammed 3’ CpG Island Methylation Confers Tissue- and Cell-Type-SpecificTranscriptional Activation. Molecular and Cellular Biology 33: 1845–1858.

4. Butler MG. (2009) Genomic Imprinting Disordersin Humans: A Mini-Review. Journal of Assisted Reproduction and Genetics 26:477–486.

5. Bachman et al. (2014) 5-Hydroxymethylcytosineis a Predominantly Stable DNA Modification. Nature Chemistry 6: 1049–1055.

6. Raiber et al. (2015) ​5-Formylcytosine Altersthe Structure of the DNA Double Helix. Nature Structural and Molecular Biology22: 44–49.

7. Ito et al. (2011) Tet Proteins can Convert5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine. Science 333:1300-1303.

8. Song et al. (2013) Genome-wide profilingof 5-formylcytosine reveals its roles in epigenetic priming. Cell 153: 678–691.