for easy SNP and mRNA mapping with subcellular resolution. In
the present chapter, we exploit a SNP within conservedACTB
mRNA to discriminate human and mice cocultured cells and
apply described protocol to genotypePCDH11homologs in pre-
served brain tissue.2 Materials
2.1 RT Primers 1. 100 μM p_hsa_ACTB b 5^0 -C+TG+AC+CC+AT+GC+CC
+ACCATCACGCCC.
- 100μM p_mmu_ACTB b 5^0 -C+TG+AC+CC+AT+TC+CC
+ACCATCACACCC.
+¼LNA-modified base.
For mRNA genotyping, we highly recommend using LNA
modified primers [9] to maximize RT rate (random decamers can
be used as universal RT primers, however the RT efficiency is lower
probably due to lower hybridization strength). Primer should be
designed in accordance with standard PCR primer design instruc-
tions (only a single, “reverse” primer is required for in situ RT).
Primers should be 20–25 bp long, ~50% GC content and mini-
mized probability of forming 3^0 hairpins, homodimer or hetero-
dimer. Online tools like primer3 [12] are a great start as they
suggest the best “right” RT primer with all thermodynamic para-
meters accounted for. LNA substitutions have to be suggested by
the researcher and we typically substitute 3–7 DNA bases with LNA
derivatives (seeNote 2). Primer hybridization site should be down-
stream (3^0 ) and as close to the SNP as possible (seeNote 3and
Supplementary Note 3 in [13]). Once LNA primer sequence is
determined, it should be evaluated for potential off target RT
priming to prevent formation of false-positive cDNA and signals.
Blastn against RefSeq mRNA database is recommendedhttp://
blast.ncbi.nlm.nih.gov/Blast.cgi. Figure 2 shows the ACTB
mRNA in human and mouse with SNP highlighted, illustrating
typical design example for both LNA primer and allele specific
PLPs.2.2 Padlock Probes
and Decorator Probes
- 2 μM hsa_ACTB 5^0 GCCGGCTTCGCGGGCGA
AAAAAAAAAAA CCTCAATGCACATGTTTGGCTCC
AAAAAAAAAAAACGGCGCCGGCATGTGCAAG. - 2 μM mmu_ACTB 5^0 GCCGGCTTCGCGGGCGA
AAAAAAAAAAA CCTCAATGCTGCTGCTGTACTAC
AAAAAAAAAAAACGGCGCCGGCATGTGCAAA.
Underline¼target complementary arms, italic¼detection
probe complementary sequence.
212 Tomasz Krzywkowski and Mats Nilsson