Sure, here are 10 key points about microRNA (miRNA): 1. **Definition**: miRNAs are small non-coding RNA molecules, typically about 22 nucleotides long, that regulate gene expression post-transcriptionally. 2. **Gene Regulation**: They play crucial roles in regulating gene expression by binding to complementary sequences on target messenger RNA (mRNA), leading to mRNA degradation or translational repression. 3. **Biogenesis**: miRNAs are transcribed from DNA into primary miRNA (pri-miRNA) transcripts by RNA polymerase II. These transcripts are processed into precursor miRNAs (pre-miRNAs) by the Drosha enzyme complex in the nucleus, and further processed into mature miRNAs by the Dicer enzyme complex in the cytoplasm. 4. **Function**: miRNAs are involved in various biological processes such as development, differentiation, apoptosis, and metabolism. They can act as either oncogenes or tumor suppressors depending on the context. 5. **Target Recognition**: miRNAs typically bind to the 3' untranslated region (UTR) of target mRNAs through base-pairing interactions, although they can also target other regions. 6. **Disease Implications**: Dysregulation of miRNA expression has been linked to various diseases including cancer, cardiovascular disorders, neurodegenerative diseases, and immune disorders. 7. **Therapeutic Potential**: miRNAs have emerged as promising therapeutic targets or tools for treating diseases. miRNA-based therapeutics include miRNA mimics, antisense oligonucleotides, and small molecule modulators. 8. **miRNA Profiling**: miRNA expression profiling is widely used in research and clinical settings to study disease mechanisms, identify biomarkers, and develop diagnostic or prognostic tools. 9. **Evolutionary Conservation**: miRNAs are evolutionarily conserved across species, highlighting their fundamental roles in gene regulation and development. 10. **Technological Advances**: Advances in high-throughput sequencing and bioinformatics have greatly facilitated the discovery and characterization of miRNAs, enabling a deeper understanding of their functions and mechanisms of action.

20. • Primer designing
• Primer is a short stretch of sequence that serves as an
initiation point for DNA synthesis. There can be a set of
primers (forward and reverse) with a sequence
complementary to the template DNA -a point of initiation
synthesis.
• The main objective of the primer is synthesizing
DNA with a free terminal end and initiation point of
polymerase.
• The forward primer runs in 3’-5’ while the reverse primer
runs in 5’-3’. However process of elongation results in
two new strands of ds DNA.
• There are two types of primers
• Namely- DNA Primers and RNA Primers.

21. • DNA primers are long-lived and more stable while RNA primers are
short-lived and are more.
• Primer Designing Rules:
• Primer length: Oligonucleotides between 18-24 are said to be quiet
enough and advantageous so that short primers would bind easily to
the template at the annealing temperature.
• Melting temperature (52°C-56°C) The GC results of the sequence
gives a fair indication of the primer Tm. However, the difference of
the primer should not be less than 2°C.
• Primer Annealing (Ta): The high Ta results in low PCR product with
insufficient primer-template hybridization, while too low Ta will lead
to non-specific PCR products caused as a result of a high number of
base pairs mismatches.
• Ta= 0.3*Tm (primer) +0.7 (product) – 14.9, Tm (primer)
• Melting Temperature of the Primer:
• Tm (primer)- It measures the least stable primer-template pair.
• Tm (product)- It measures the melting temperature of the PCR
product.
• The modified step annealing can be performed using gradient PCR
where temperature can be set to bind primers.

22. • Tm: 4(G+C)+2(A+T) 0C
• Primer GC content and Clamp: Gene sequencing Primers
must possess GC content between 40-60%, with the 3’ end,
by with 2 GC bases- GC clamp. However, GC bp with 3 H
bonds that are stronger than AT bonds with 2 bonds with the
high stability of the primer along with the improvement and
specificity of the primer binding.
• Setting Restriction Enzyme(RE) Cut Sites: The enzyme
called leader sequence permits the higher efficiency for
cutting enzymes by adding 3-5 bases to the 5’ end of the
total cut site in our target primer.
• End Stability: The maximum G allows the binding of 3-5
least bases with the 3’ end. However, a stable 3’ end can
reduce false priming.

23. • Caution for designing PCR Primers:
• Hairpins: The loop structure formed by the
intramolecular interactions within the primer which
optimally 3’ end with -2kcal/m and internal hairpin with -
3kcal/m can be tolerated.
• Dimers: A structure forming ds DNA by intermolecular
interactions between 2 primers. Likewise, if the
interaction formed between 2 homologous or the same
sense of primer, – called as self-dimers while the opposite
primers are called as cross dimers.
• Repeats & Run: The consecutive occurrence of
dinucleotide runs in the continuous stretch of a single
nucleotide is considered the most important property. The
maximum no. of repeats and runs was of 4 dinucleotides
and 4 base pairs.

24. • Primer- Template Cross Homology: Primers should be
designed in such a way that no homology within the
template is been noticed other than the target site which
resulted in non-specific binding and amplification. This can
be categorized into 2 types: a) Intra-primer
homology: The complementary bases within the same pair
in the region of more than 3 bases can cause intramolecular
bonding b)Inter-primer homology: Forward and reverse
primers with complementary sequences are responsible for
intermolecular bonding.
• Soft wares for Primers designing
• Primer3 Input
• Primer3Plus (bioinformatics. NL)
• PrimerQuest – design qPCR assays | IDT (idtdna.com)
• PerlPrimer (sourceforge.net).