ACBB Biotechnology Project

CMB: Neurobiological track [Digitare qui] ACBB BP 22/23

ACBB Biotechnology project

Sequence alignment 17/10/2022

Neuregulin-1 (NRG1) is a ligand of the ErbB receptor family. ErbB receptors are tyrosine kinase receptors and they include 4 different types: ErbB1, ErbB2, ErbB3 (only heterodimers) and ErbB4 (heterodimers or homodimers). NRG1 receptors are ErbB3 and ErbB4.

NRG1 can either be soluble (important for autocrine and paracrine signalling) or transmembrane. It plays different roles in different tissues, including myelination, neuronal migration during development and migration to the olfactory bulb in adulthood, tangential and radial migration of neurons, synapse formation, neuromuscular junction (NMJ) formation, oligodendrocyte development and even in brain and breast tumours.

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The transmembrane NRG1 can have 3 different alternative splicing isoforms: type I, type II, and type III. NRG1-type III has a transmembrane domain (indicated as TM) on its first exon, thus this isoform type has an extra transmembrane domain on top of the other one that has in common with all three isoforms. After the first exon (type-specific sequence), each of the three NRG1 isoforms has different domains, such as the Ig-like domains (Iga and Igb) and the two spacers S1 and S2 in type I and type II, but all three types share a common domain, the EGF-like domain. The EGF-like domain is the one binding to the ErbB receptor, and it is followed by either the α or β domain. The β domain is then followed by either one of 3 different domains: 1, 4 and 3*, where the * after 3 indicates the presence of a STOP codon. The 1 and 4 domains are then followed by a cytoplasmic domain (TMc) that contains a transmembrane domain followed by domains a*, *c or b*. Again, here the * indicates the presence of a STOP codon.

So in all isoforms, meaning type I, type II and type III, there is the EGF-like domain, and all three can have the α or β domain followed by a transmembrane domain, or all three isoforms can have the β 3* domain and thus terminate without the cytoplasmic (TMc) domain.

Interestingly, NRG1 types that have the β domain have a stronger ability in activating signal transduction and are expressed by different cells.

Type I and type II isoforms will give rise to transmembrane proteins as well as soluble proteins. Type III isoforms will give rise to proteins with a single transmembrane domain (β-3* proteins), or proteins that cross the membrane twice. All types can be proteolytically cleaved and give rise to soluble proteins following stimulation. The type III β-3* can is ready to interact with the ErbB receptor, without the need for proteolytic cleavage.

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Comparing different sequences by means of alignment tools is useful to identify different domains and thus classify the NRG1 type (e.g. III I-α1a, II-β1a) that is being investigated. β-3*,

In the genome, each type-specific domain (= the first exon) is preceded by a promoter, and each species has a different organization of the constituting domains that gives rise to different combinations of each NRG1 type.

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Placing primers on rat NRG1 isoforms

In order to identify/quantify the expression of α and β isoforms regardless of the NRG1 type, the forward (sense) primer for RT-PCR must be placed on the ATG of the EGF-like domain since it is the only domain common to all three isoforms, and the reverse (antisense) primer must be placed on the STOP codon of the α domain to identify the α isoform, whilst it must be placed on the STOP codon of the β domain to identify the β isoform.

In order to identify type I, type II or type III, the forward (sense) primer for RT-PCR must be placed on the ATG of each type-specific sequence (= the first exon), whilst the reverse (antisense) primer must be placed on the STOP codon of the EGF-like domain.

In order to identify all NRG1 isoforms both forward and reverse primers must be placed on the EGF-like domain, since this is the domain that all three isoforms have in common and it is not specific to each one of them.

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In order to clone the entire III β-3 cDNA and thus have the expression of the full length NRG1 III-β-3 protein, the forward (sense) primer must be placed before the ATG of the type III specific sequence (= type III first exon), whilst the reverse (antisense) primer must be placed after the STOP codon on the β 3* domain.

5’ UTR and 3’ UTR

The UTR acronym stands for untranslated region, in fact, these are the nucleotides preceding the ATG of the coding sequence (CDS) in the case of the 5’ UTR, and the nucleotides after the STOP codon of the CDS.

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Designprimer 24/10/2022

To study gene expression, primers are prepared and usually real-time PCR is performed afterwards.

Primers are always in pairs: a forward (sense) primer (its sequence matches the sense DNA sequence, i.e. the upper strand 5’ -> 3’) which binds its 5’ to the 3’ of the ANTISENSE (bottom) DNA strand, and a reverse (antisense) primer which binds its 5’ to the 3’ of the SENSE (upper) DNA strand, following DNA denaturation. This occurs because the forward (sense) primer sequence is complementary to the sequence of the ANTISENSE (bottom) DNA strand, whilst the reverse (antisense) primer sequence is complementary to the sequence of the SENSE (upper) DNA strand.

The forward (sense) primer anneals (attaches) to the ATG (START codon) of the ANTISENSE (bottom) DNA strand, whilst the reverse (antisense) primer anneals (attaches) to the STOP codon of the SENSE (upper) DNA strand. Note that sometimes the first ATG is not the correct one to attach the forward primer.

Never forget that the DNA-pol READS the DNA strand in the direction 3’ à 5’, and SYNTHESIZES the new DNA strand in the direction 5’ à 3’.

Primers must always be written 5’ à 3’. Remember that when writing the reverse primer you start READING from the 3’ end, and as you move right to left (ß 3’), you write the bases left to right (à 3’).

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Oftentimes, it is hard to find where the 5’ and the 3’ are located, so remember that the upper DNA strand is always 5’ à 3’.

The length of a primer is dependent on the sequence of the primer itself, since it is the sequence that determines the melting temperature of the primer. The melting temperature (Tm) is the temperature at which 50% of the primer will have annealed to the DNA strand. Each A or T that anneals to the DNA strand has a melting temperature of 2°C, whereas each C or G that anneals to the DNA strand has a melting temperature of 4°C. The ideal melting temperature for primers is 60°C (59°C-61°C): if the Tm is <60°C the primers will form secondary structures with each other or themselves, if the Tm is >60°C the majority of the primers will detach from the DNA strand.

Primer pairs must meet the following criteria:

• Similar Tm of approximately 60°C (according to Allawi: 59°C-61°C).
• 18-30 nucleotide long.
• End with either G or C à G or C at their 3’ end.
• Content of G and C (GC%) of ≥50% à dictates the length of each primer.
• Not form secondary structures such as dimers or hairpin loops (<4 consecutive pairing indicated as I on Annhyb) neither with themselves nor with each other.
• Placed in such a way that the amplification product will be 80-120bp long.

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Where to put primers

To amplify and clone the full-length cDNA to express the corresponding full-length protein, the forward primer is placed before ATG and the reverse one after the STOP codon. This placement is essential for the ribosome to recognize the beginning and the end of the mRNA so that they can properly translate it into protein.

To verify/amplify the expression a gene/specific isoform, it is enough to amplify a short region of DNA and not the entire coding sequence (CDS), as this will ensure 100% efficiency in real-time PCR. Therefore, it is more appropriate to place both primers on one exon, of course one at the ATG and one at the STOP codon.

However, during mRNA extraction and purification it is possible to have some genomic DNA (gDNA) contamination, which unfortunately will be amplified during real-time RT-PCR along with the cDNA resulting from mRNA reverse transcription. Moreover, gDNA contamination can alter the results of the study, as some samples might more contaminated than others, or it can interfere with the correct interpretation of the results if it is being investigated whether the expression of a gene/protein is affected by the treatment.

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Usually, this issue is overcome by performing DNAse treatment, however, if the RNA extracted is too small it is more complicated. Luckily, there are a few strategies to ensure only cDNA is amplified and not the contaminant gDNA.

Strategy #1: Design primers on different exons that are separated by an >1000bp long intron. If performing end-point RT-PCR it is better if the intron is >2000bp long rather than 1000bp. By doing so, the >1000bp long intron will be spliced out of the mRNA and not translated, and the >1000bp long intron in the gDNA will not be amplified by the Taq-polymerase during the short elongation period. The resulting amplification product will be 80-120bp long.

Strategy #2: If introns between the different exons are smaller than 1000bp, design primers across the two exons. This means that at least one primer will anneal on half of the first exon and the other primer will anneal on half of the second exon. By doing so, the small intron between the two different exons will not appear as a second band in end-point RT-PCR, and it will not make it hard to distinguish between the gDNA and the cDNA during quantification in real-time RT-PCR.

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What happens if there is only a single exon

Sometimes, as in the case of housekeeping genes, the coding sequence consists of just a single exon. More than housekeeping genes, coding sequences consisting of a single exon are observed in pseudogenes, which are extra copies of cDNA in the gDNA that code for the mRNA of interest. Pseudogenes are characteristics of viruses or positive selection if the gene is important for the species. Unfortunately in these cases, it is not appropriate to insert both primers on that single exon, otherwise both the gDNA and the mRNA will be amplified.

To overcome this issue, it is recommended to:

• Perform DNAse treatment.
• Use a control for the contamination.

The control that enables to detect any DNA contamination such as gDNA, plasmid DNA, amplification product contamination is the RT-negative control, which lacks the reverse transcription (RT) enzyme. If during the amplification process bands are produced in the RT-negative control, it means that there is gDNA contamination. The RT-positive sample will instead contain the RT enzyme, thus it is the sample where bands are expected.

It is also recommended to include another control when performing RT-PCR, i.e. the positive control, which consists of a tissue/cell derived mRNA that is known to be expressed.

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Therefore, for single exon experiments the RT-positive sample, the RT-negative control and the positive control are needed.

Note that random primers are added in the reaction mix, which are short primers that recognize mRNA and are necessary for the RT enzyme to retro-transcribe mRNA into cDNA.

In addition, to verify that none of the reagents in the reaction mix is contaminated by DNA (plasmid DNA or amplification products), a further control is needed: the no template control (NTC). In the NTC, no cDNA (= no template) is added, but nuclease-free water is added instead. In case a band is observed in the NTC, it means that at least one of the reagents was contaminated with DNA.

Replicates

Experiments have two different kinds of replicates: technical or biological.

Technical replicates include all the repetitions of the same technique for the same sample that are done in one day of the experiment, for example, PCR of sample 1 is repeated 3 times. It is recommended to use at least 3 technical replicates for each experiment in order to obtain valuable results.

Biological replicates, denoted at “n=…”, include all the different days in which the entire experiment is repeated in different cell lines/animals and in different conditions. Even in this case it is recommended to have a minimum of 3 biological replicates in order to have statistically significant difference when performing statistical analysis.

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Controls for RT-PCR 07/11/2022

Controls are needed not only to normalize data, as in the case of housekeeping genes, but to also verify that all the reactions and reagents worked well and there was no contamination.

The images below are results that can be potentially obtained from RT-PCR.

Note that:

• RT reaction negative control (RT-) is the control where all RT reaction reagents and RNA are included, but there is no RT enzyme.
• PCR reaction negative control is the PCR no-template control (NTC), where all PCR reagents are included, but there is no template, i.e. there is no cDNA but nuclease-free water instead.

If after PCR, there is no band in the sample (#) it means that the gene of interest is not expressed. However, if there is no PCR positive control, it is not possible to conclude that the absence of the band in the sample (#) is actually due to the experimental treatment or if the gene was absent in the first place.

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The PCR positive control is a sample where the gene of interest is known to be expressed. Therefore, it ensures that the absence of the band in the sample (#), and thus the absence of the gene of interest, is not due to PCR technical problems.

However, having PCR positive control is not sufficient to determine whether RNA was extracted well or reverse transcription properly worked, therefore a further control is needed. In this case a housekeeping gene is needed, as it is a gene that is ubiquitously expressed and thus aids in data normalization and in verifying that the cDNA was correctly and how efficiently retro-transcribed.

If there is a band representing the housekeeping gene in the sample (#) and a band in the PCR positive control, but no band representing the gene of interest in the sample (#), then it can be concluded that the absence of the gene of interest is due to the experiment and not to technical issues. In fact, the PCR properly worked as evidenced by the band in the PCR positive control, and the cDNA was correctly retro-transcribed as evidenced by the band representing the housekeeping gene in the sample (#).

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Among the numerous housekeeping genes, GAPDH is not really good because it has a single exon and pseudogenes in the genome.

The above results indicate that the gene of interest is expressed and that all the reactions properly worked as evidenced by the band in the PCR positive control and by the band representing the housekeeping gene in the sample (#).

The above results evidence the presence of 3 bands of the same molecular weight: one in the sample (#), one in the RT- control (A) and one in the PCR positive control (C). Additionally, the band in the sample (#) and the band in the PCR positive control (C) have the same intensity, whereas the band in the RT- control (A) is less intense than the other two.

This indicates that there is no contamination in the PCR mix (B) because there is no band in the PCR NTC (B), but there is contamination in the RT- control (A), which could be in the RNA or in the RT mix because of the band present despite the absence of the RT enzyme.

This contamination could be due to plasmid DNA or previous PCR where primers were placed on two different exons. It could be gDNA contamination if the molecular weight of the gDNA

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contamination was the same as that of the amplification product (band in sample #), which is caused by the primers being placed on one exon.

The above results evidence the presence of 4 bands of the same molecular weight: one in the sample (#), one in the RT- control (A), one in the PCR NTC (B) and one in the PCR positive control (C). Moreover, the band in the sample (#) and the band in the PCR positive control (C) have the same intensity, whereas the band in the RT- control (A) and the band in the PCR NTC (B) are less intense than the other two.

The presence of all these bands indicates contamination in the PCR mix (B) despite the absence of cDNA template, and contamination either in the RNA or in the RT mix (C) despite the absence of the RT enzyme.

The above results evidence the presence of 2 bands of the different molecular weight in the sample (#), the one at the bottom more intense the one on top. The upper band could be an alternative splicing isoform or an a-specific amplification product. To verify the identity of this

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upper band, the DNA in the upper band can be cut from the sample, cloned in a vector and then subjected to sequences analysis.

The above results evidence the presenc