are used for every target to allow discrimination (unless multi-
ple SNPs are to be visualized simultaneously,seeNote 8). Once
the full PLP sequence is designed we strongly advice evaluation
of probe secondary structure and off-target binding. We typi-
cally use mfold [16](http://mfold.rna.albany.edu) or OligoA-
nalyzer for secondary structure predictions. Use prediction
parameters adjusted for ligation reaction conditions (seeNote
5 ). Every change in PLP sequence (different decorator motif or
linker sequence) should be followed by a secondary structure
prediction (seeNote 9). To prevent formation of false-positive
signals PLP target sequence (cDNA) should be blasted (http://
blast.ncbi.nlm.nih.gov/Blast.cgi.) against refseq mRNA
database.- 10μM hsa_DO 5^0 CCTCAATGCACATGTTTGGCTCC.
- 10μM mmu_DO 5^0 CCTCAATGCTGCTGCTGTACTAC.
Decorator probes (seeNote 8).
2.3 Reagents All enzymes should be stored at^20 C. Other reagents are stored
at room temperature (RT) unless stated different.
- 40 U/μL RNase Inhibitor (e.g., DNA Gdansk
RIBOPROTECT). - TRANSCRIPTME—RnaseH-reverse transcriptase 200 U/μL
and buffer (DNA Gdansk,seeNote 10). - 40 U/μL Tth DNA ligase (e.g., GeneCraft).
- 5 U/μL RNaseH.
- 10 U/μL Phi29 DNA polymerase and buffer (e.g., Thermo
Scientific). - Biological sample: cultured cells (alive) or tissue (fresh or fresh
frozen). Fixed or freshly frozen tissues should be stored at
80 C. - Diethylpyrocarbonate (DEPC). Stored at 4C(seeNote 11)
Known carcinogen. Work under a fume hood, wear gloves. - RNase and DNase inactivators.
- 70, 85, and 99.5 v/v % ethanol.
- Xylene.
- 0.1 M hydrochloric acid.
Highly corrosive. Work under fume hood with rubber PVC gloves
and eyes protection. - 3.7% formaldehyde in PBS (seeNote 12).
Known carcinogen. Use nitrile gloves, eyes protection and handle
powder in the chemical fume hood. - Pepsin, lyophilized powder, 2.500 U/mg protein (seeNote 13).
- 0.25%trypsin–EDTA.
214 Tomasz Krzywkowski and Mats Nilsson