br Breast tissue collection in this work was approved
Breast tissue collection in this work was approved by the Ethics Committee of Nanjing Medical University. All subjects gave written informed consent before taking part in the study. Paired samples of tumors and adjacent normal tissue were obtained from 102 breast cancer patients, with consecutive collection from January 2011 to December 2011 at the First Affiliated Hospital of Nanjing Medical University and Nanjing Drum Tower Hospital, Nanjing, China. The patients age ranged from 38 to 64 years, mean patient age: 51.9 ± 8.9 years. The diagnosis of each case were independently confirmed by two pathologists based on WHO classification. The clinical stage was classified in accordance with the American Joint Committee on Cancer (AJCC) tumor-lymph node-metastasis (TNM) classification system. Immediately after surgery, tissue samples were stripped of adhering fat and cut into small pieces, frozen in liquid nitrogen and stored at 80 C. Before RNA isolation, they were first thawed to room temperature, then rinsed thoroughly with DEPC water. Total RNA isolation from G 418 or approximately 50 mg tissue homogenates was conducted by using RNAiso Plus. RNA concentration was estimated with a Thermo nanodrop spec-trophotometer. The RNA extracts were stored at 80 C until further processing. In addition, ER, PR and HER2 of the stored samples were also evaluated. The HER2 status was evaluated ac-cording to the ASCO/CAP guidelines and only a 3 þ HER2 score was considered as positive, and the ER and PR positive cut-off points were deemed positive if >1% of tumor cells were stained.
2.3. Preparation of DPD probe
Prior to conjugation, the synthetic DNA containing a 3’ end di-sulfide modification needed to be reduced. Briefly, 4 nmol DNA was first reduced utilizing 300 mL TCEP reducing beads. After 1000 g centrifugation for 5 min, the supernatant was collected. Then, 300 mL of 100 nmol maleimidohexanoic acid-modified peptide dendrimer was added to the solution. The conjugation reaction was carried out at 37 C for 4 h prior to purification. The DPD compound
Fig. 1. Peptide dendrimer containing eight reporter peptides and tryptic cleavage sites was first ligated to a DNA sequence at the 50 end, which was complementary to that of the target miRNA. Afterward, the newly synthesized DPD probe was hybridized with the miRNA that was biotinylated at the 30 end and bound to streptavidin agarose through streptavidin-biotin interaction in advance. After the treatment with trypsin digestion, the reporter peptide was released and quantified using LC-MS/MS. In this way, the assay sensitivity can be enhanced by signal amplification.
was separated from the excessive peptide dendrimer by high per-formance liquid chromatography (HPLC) (please see the supple-mentary material). Finally, the collected fraction was ultrafiltered with Amicon Ultra 3K device (Merck Millipore, Darmstadt, Ger-many). Quantification was carried out with external calibration peak area measurement.
2.4. miRNA hybridization with DPD probe
Immobilized miRNAs (Please see the supplementary material
for miRNA biotinylation and immobilization) was incubated with
. After hybridization, the beads were thoroughly washed to
avoid nonspecific binding. In the present work, optimization of
hybridization temperature and time were done. For the purpose of
evaluating the hybridization specificity of probe, miR-21 molecules with single-base mismatch (50-UAGCUUAUCAGUCUGAUGU-UGA-
30) and two-base mismatch (50-UAGCUUAUCAGUGUGAUGUUGA- 30) were also examined.
2.5. In-solution tryptic digestion
The above solution containing miRNAs: DPD hybrid was mixed with 90 mL of 50 mM NH4HCO3. Afterward, trypsin (sequencing-
grade) was added to the sample and then incubation at 37 C for 24 h with shaking. Thereafter, the reaction was inhibited by adding 10 mL of 0.1% TFA. After that, 100 mL internal standard solution was added to the solution, then the digestion mixture was transferred into a microspin C18 column (The Nest Group, Inc., MA, USA). Prior to loading, 100 mL ACN and 100 mL water was added into the column for pretreatment. Then the column was washed with 50 mL of 5% ACN (0.1% TFA), and after that, 50 mL of 80% ACN (0.1% FA) elution buffer was added to the column. The elution cycle was repeated 3 times. For detailed information, please see the manufacturer's operating instruction.
Samples were analyzed on analytical platform: Agilent Series 1290 UPLC system (Waldbronn, Germany) coupled to an Agilent 6460 Triple Quad LC-MS mass spectrometer (Santa Clara, USA) which was equipped with an electrospray ion source. Mobile phase A was composed of water, 0.02% formic acid. Mobile phase B was composed of methanol, 0.02% formic acid. Flow rate was 0.3 mL min 1. An Agilent SB C18 (2.7 mm, 30 mm 2.1 mm) was used for chromatographic separation. The peptide sample was separated with a 10 min LC gradient starting with 10% B, keeping isocratic condition for 1 min, then increasing to 90% in 3 min, and holding for another 4 min and then, back to initial conditions within 1 min. The column was re-equilibrated for 2 min before