Molecular Biology C
Conception, theory, research, and application
——Logic and LIY (Learn It Yourself)

Sheng Zhao (赵晟), Biochemistry and Molecular Department of Medical school in
Southeast University

Couse QQ Club: 112342994 (分子生物学C )

Web: http://teaching.ewindup.info/

Email: shengzhao@seu.edu.cn or windupzs@gmail.com

QQ /MSN/Skype/gChat: windupzs@gmail.com

Mobile:18551669724 or 13675130010
Chapter 4: Finding Scientific Nemo (From
clinic samples to scientific research)
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)
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
• Acute and convalescent serum specimens
• Whole blood for Culture
• Whole blood plasma for molecular biology
• Aliquot sera
• Collection of Tissue Specimens
• Fixed tissues
• Complete autopsy on all fatal cases
• Non-fixed tissues
Target population
INPATIENTS
Lower Respiratory
OUTPATIENTS
• Bronchoalveolar lavage,
Upper Respiratory
• Nasopharyngeal (NP) tracheal aspirate, pleural
fluid
and oropharyngeal
• Sputum
(OP)
• Nasopharyngeal
Upper Respiratory
wash/aspirate
• Nasopharyngeal (NP) and
oropharyngeal (OP) swabs
Lower Respiratory
• Nasopharyngeal wash /
• Sputum
aspirate
Blood
Blood
• Serum: Acute (at
• Serum: Acute (at onset)
onset) and
and convalescent (3-6 weeks
convalescent (3-6
post onset)
weeks post onset)
• Whole blood (plasma)
• Blood (plasma)
• Urine
• Stool
• 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
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
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
1
Double
n cycles
(2)n
2
3
Linear plot
Ideal outcome
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
1400000000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
30
35
30
35
PCR CYCLE NUMBER
Log plot
AMOUNT OF DNA
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
AMOUNT OF DNA
1600000000
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
5
10
15
20
25
PCR CYCLE NUMBER
Only in the exponential phase, PCR product depends on
template
Why endpoint analysis doesn’t work
Out of exponential phase
SERIES OF 10-FOLD DILUTIONS
False amplification (Blank)
Need enough initial template
2500
2000
1500
1000
500
0
1
4
7
10
13
16
13
16
19
22
25
28
31
34
37
40
43
46
49
8
7
6
5
4
3
2
1
0
1
4
7
10
19
22
25
28
31
34
37
40
43
No enough reaction volume (=10 ul), CV%>3%
4.00E+03
3.50E+03
3.00E+03
2.50E+03
2.00E+03
1.50E+03
1.00E+03
5.00E+02
0.00E+00
1
4
7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
Alignment of the machine
Treat the machine as a baby, gently please.
Example: Determine quality of experiments
7
2.8
R a w flu o re s c e n c e
R a w flu o re s c e n c e
6
5
2.6
E=0.562
E = 0 .8 0 3
2.4
4
2.2
0
4
8
12
16
20
24
28
32
36
40
44
48
C ycle n u m b er
3
E = 0 .5 6 2
2
0
4
8
12
16
20
24
28
C y c le n u m b e r
32
36
40
44
48
Melting curve to valuate primer