Bead-bait hybridization assay

At each assay step, the beads were mixed by vortexing for 10 s every 2 min to avoid sedimenting. A biotinylated capture probe (0.2 nmol) was added to streptavidin-coated magnetic beads (50 µL) in 1X PBS. After 10 min, the supernatant was removed, and the beads were washed twice with 1xPBS (100 µL). An RNA sample (20 ng/µL; 5 µL) was added, and the mixture was kept at room temperature for 20 min, followed by removing the supernatant and washing the beads (2× 1X PBS, 100 µL). The linker probe (0.2 nmol) was added for 20 min at room temperature, followed by three washes at elevated temperature (100 µL for each wash with buffer pre-heated to 42 °C) and adding ct DNA (2 mg/mL, 5 µL) for 20 min at room temperature.

Linker probe sequences were designed to be located 20–50 nucleotides upstream to mutation-specific capture probes, as described⁵⁵, and they were as follows:

BRAF, 5′-d(A+TC AG+T TTG AAC A+GT+TG TTTT GA+T GG+G AAT A+CC AGA C+CA C+CTG)

KRAS, 5′-d(A+CG A+AT ATG ATC+CAA C+AA TTTT GA+T GG+G AAT A+CC AGA C+CA C+CTG)

EGFR, 5′-d(A+TA+TAT AAT GTG A+CT T+CA TTTT GA+T GG+G AAT A+CC AGA C+CA C+CTG)

where LNAs are indicated with a plus in front of the corresponding nucleotide; the sequence part binding to ct DNA is shown in bold. Ct DNA genome accession code used for the linker sequence design was {"type":"entrez-nucleotide","attrs":{"text":"NC_037330.1","term_id":"1378962611","term_text":"NC_037330.1"}}NC_037330.1, GI: 1378962611. The part of the linker probe shown in bold has a higher affinity to a ct DNA (+) strand than its complement. Therefore, a linker probe acts both as target RNA-binding reagent and as invader probe to ct DNA.

The supernatant was removed and after four washes (100 µL 1X PBS); then we added 7% DMSO in 1X PBS (50 µL), which denatured the duplex⁴⁷. The supernatant was removed and recovered in 1X PBS using an Amicon 3 MWKO device (Millipore). The final volume of the recovered sample was 10 µL; 0.6 µL Eva Green (Biotium, 20X stock) fluorophore was added and the fluorescence was measured immediately using a 384-well plate Roche Light Cycler 480 plate reader. The data was analysed using Light Cycler fluorescence analysis software following the manufacturer’s recommendations.

Reverse transcription (RT) and LNA/DNA PCR primers were synthesized by Qiagen following the previously reported considerations^45,46. Per PCR reaction, we used 100 ng RNA extracted from cell lines with an average length of 1.1 kb, as determined by a Qiasymphony SP instrument.

Reverse transcription was carried out as follows: mix and incubate at 42 °C for 1 h the following components: RNA target (5 µL; 20 ng/µL), the corresponding RT primer (50 µM; 2 µL); RT supermix (Qiagen) (10 µL); and 3 µL RNAse-free water (20 µL total volume). The enzyme was inactivated at 85 °C for 5 min. Each experiment has been carried out in triplicate.

Reverse transcription gene-specific primer sequences were as follows:

BRAF, 5′-d(AGA GCT CTT ATC AAT TTG TTG CAA CGA AC)

EGFR, 5′-d(AGA GCT AGT ATA GAG GTC TTA CAC ATT TTT GT)

KRAS, 5′-d(AGA GCT ACT TTA TAA GCC ATA GAC ACT ATA GT)

Upon RT completion, DNA was purified by an Amicon device, MWKO 100 kDa, and reconstituted into ultra-pure water to a total volume of 5 µL (Millipore/Sigma Z648043). PCR was carried out immediately using the following primer pairs:

LNA-DNA PCR primers:

BRAF, Forward 5′-d(GCC TGA AGA CCT CAC AGT AA); Reverse 5′-d(ACT CCA TCG AGA TTT C+T);

KRAS G12D, Forward 5′-d(GTG GTA GTT GGA GCT G+T); Reverse 5′-d(AGA GTG GCC CTT GAC GAT ACA).

EGFR L858R, Forward 5′-d(GCA TGT CAA GAT CAC AGA TT); Reverse 5′-d(CCA GAC CCA AGT TTG GCC C+T), LNA is marked with a plus in front of corresponding letter. Nucleotides opposite to the mutation in the target are underlined.