Molecular Biology C SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University CCouse QQ Club: ( 分子生物学 C ) WWeb: E or QQQ /MSN/Skype/gChat: MMobile: or Conception, theory, research, and application ——Logic and LIY (Learn It Yourself)

Section 1 : Trust it or trash it? ——Number, selection, and target (Establish sample library) Section 2 : A colorful microscopic world ——Specific labeling (Visualization techniques) Case 4 : Immortal clinic samples ——Primary culture and gene manipulation (Construction of the cell line) Chapter 4: Finding Scientific Nemo (From clinic samples to scientific research)

Sample library

Setup your library Define your goal Question based Type of study Time frame Involved techniques Planning for specimen collection Specimen collection and processing Storage, packaging, and transport of specimens Performing experiments, analysis, and publication Request more specimen

Planning for specimen collection Always plan in advance 1.Who 2.Where 3.When 4.What 5.How Define the possible questions you need to answer which clinical specimens are required to answer these questions Plan for the possible scale of the study Decide how to collect, process and transport the specimens Define the procedures necessary for specimen management Biosafety issues

Specimen collection and processing Safety and decontamination procedures Basic safety precautions Gloves, clothing, mask, etc. Handle needles 1% household bleach for soak, 10% for clean up spills Awareness Labelling and identification of specimens Preprinted labels (at least five) should be used whenever possible. patient name unique identification number specimen type and date and place of collection name or initials of specimen collector. Label accompanying forms Aliquots!

Storage, packaging, and transport Storage  Aliquots!!  Freezer (e.g. -80C)  Liquid nitrogen Packaging Blue ice Dry ice Transport Multicenter clinical study Notify the receiving laboratory Car, High speed rail way, Flight, etc.

Examples 1. Collection of Respiratory Tract Specimens Upper Respiratory tract Optimal timing are usually earlier Swabs, Nasopharyngeal aspirates, Nasal washings, Oral cultures, etc. Lower Respiratory Tract Specimens Less time sensitive Tracheal aspirate, sputum, pleural fluid, Open lung biopsy, etc.

Examples 2. Blood and tissue specimens Collection of Blood Components Collection of Blood Components Acute and convalescent serum specimens Whole blood for Culture Whole blood plasma for molecular biology Aliquot sera Collection of Tissue Specimens Collection of Tissue Specimens Fixed tissues Complete autopsy on all fatal cases Non-fixed tissues

Target population OUTPATIENTS Upper Respiratory Nasopharyngeal (NP) and oropharyngeal (OP) Nasopharyngeal wash/aspirate Lower Respiratory Sputum Blood Serum: Acute (at onset) and convalescent (3-6 weeks post onset) Blood (plasma) Urine Stool INPATIENTS Lower Respiratory Bronchoalveolar lavage, tracheal aspirate, pleural fluid Sputum Upper Respiratory Nasopharyngeal (NP) and oropharyngeal (OP) swabs Nasopharyngeal wash / aspirate Blood Serum: Acute (at onset) and convalescent (3-6 weeks post onset) Whole blood (plasma) Tissue (e.g., lung) Urine Stool FATAL CASES All available premortem Specimens Tissue Fixed tissue from all major organs (e.g., lung, heart, spleen, liver, brain, kidney, adrenals) Non-fixed tissue from lung and upper airways (e.g., trachea, bronchus) Lower Respiratory Bronchoalveolar lavage, tracheal aspirate, pleural fluid Sputum Blood Serum Blood (plasma) Deep lung swab for bacterial culture

Experiments Select criteria to choose specimen for analysis Maximize the usage of the specimen  Convert to re-generable library  Multiple experiments Normalize among samples Feedback to collectors Request more or refined samples Or stop collect samples

Convert to re-generable library In vitro library  Sequencing library  cDNA library  miRNA library  Non-coding RNA library  Genome library Microbe library  Fungus  Bacteria  Virus Cell lines (Case 4)

Maximize the usage of the specimen High throughput methods High throughput ELSA 2D-gel and Mass mapping Next generation sequencing High content screening Sensitivity of the technology Real-time PCR Western blot Immunochemistry Pathology staining Reliability of the technology Make sure you understand the principle!

Maximize the usage of the specimen

Question: How to detect the expression for my gene(s) of interest? 1)Translation level: Western blot (density measurement, need good antibody, saturation) 2)Transcription level: Northern blot (density, saturation); RNase protection assay (density, saturation) In situ hybridization (density, saturation); Competitive RT-PCR (end point analysis) Quantitative RT-PCR (Method of choice)

PCR reaction Double n cycles (2) n

Linear plot Log plot Ideal outcome

SERIES OF 10-FOLD DILUTIONS Only in the exponential phase, PCR product depends on template Why endpoint analysis doesn’t work Out of exponential phase

False amplification (Blank) Need enough initial template

No enough reaction volume (=10 ul), CV%>3%

Alignment of the machine Treat the machine as a baby, gently please.

E=0.562 Example: Determine quality of experiments

Melting curve to valuate primer