Chapter 2 Research experiment ＆ design
2.10 Real Time – PCR principle
2.10.1 Relative Quantitation
One simple method for looking at relative differences in expression is the use of the relative CT approach. This approach employs the difference in the CT values obtained for two different sets of samples. Since CT values are obtained during the exponential phase of PCR, it is assumed that at each cycle the number of products are doubling. Assuming that the CT value is reflective of the initial starting copy, a difference of one.
Fluorescence systems used in Real-time-PCR. Systems employ either afluorescent dye such as SYBR Green (S) or employ a fluorescent probe that contains a reporter (R) and a quencher (Q) fluorochrome. Separation of the quencher from proximity to the reporter enables the
fluorescence of the reporter to be measured. We use SYBR Green-490 in our experiment.
Fig 15.Real-time PCR CT analyse principle
Fig 16. SYBR Green formula
2.10.2 Real-time PCR protocol
Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs.
Electrophoresis:
The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole:
2.11.1 DNA gel protocol
1. Add the appropriate volume of 1xTAE to a sterile container 2. Add the appropriate amount of agarose
3. Heat in a microwave with the lid slightly loosened until boiling (about 2 minutes on full power) 4. Allow to cool to 'hand warm' (50-60。C) e.g. by running under a cold tap
5. Add the appropriate amount of ethidium bromide . 6. Pour the gel into the gel casting tray
7. Remove any bubbles in the gel
8. Allow the gel at least 40 minutes to set (or less if put in a fridge) ensuring that the gel casting tray is level and undisturbed
9. When the gel has set remove the combs and casting gates and transfer to the gel tank 10. Mix 5 volumes of PCR product with 1 volume of 6x gel loading buffer
11. Load samples into the wells in the gel
12. Load 1kb ladder into at least one well in each row
13. Run the gel at the appropriate voltage for about 30 minutes 14. Photograph the gel under ultraviolet light
Fig 17. electrophoresis principle
Chapr 3
Result ＆ Discussion 3.1 C.elegans behavior
Use a kind of new quantitative way in the experiment that C.elegans behavior observes Op 50 Ecoli is scribbled on agar plate, because it shed the orbits of sports when C.elegans creeps.
We use very small graph paper cushion under agar plate.We time 1 min to calculate the total length of orbit that C.elegans creep at this moment. Because we find a simple fact in the past research of our laboratory. The magnetic field could reduce the C.elegans crawling the speed.
The following charts explain observation of C.elegans' behavior
We use N2 C.elegans. We observed the C.elegansto creep and slackening. Divided into four groups as follows
C.elegans strain additive nanoparticles culture environment
N2 wild-type no 96 hours
N2+MF wild-type no 96 hours with continued magnetic field
N2+Fe3O4 Wild-type Fe3O4 6-10nm 96 hours
N2+Fe3O4+MF wild-type Fe3O4 6-10nm 96 hours with continued magnetic field
Table 2. C.elegans behavior Classification
Fig 18. C.elegans behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05 .We find the magnetic field has lasting influence on C.elegans. The magnetic field makes the behavior slacken. Add magnetic nanoparticles increase the disparity of influencing. Assigning to four groups is for proving the prescroption and nanoparticle of the magnetic field act on together. There is an influence on influence on the magnetic field in the C.elegans for a long time
For example 2 groups 1.N2 and N2+MF
1.N2 slows down, after culturing 96 hours with continued magnetic field.
If we recorded with magnetic field, we have a fack that N2 slow down acutely . There is transient influence on influence on the magnetic field in the C.elegans during our experiment. when we take magnetic field off, the transient influence disappear. However, the long influences on slowing down still exist. The result is exciting. We add super-paramagnetic nanoparticles in our experiment. The result changes greater
3.1.1 Video recorded
We use the record system made by oneself to analyse the behavior of the C.elegans.The original text one is a video. In order to appear convenient, we use and pursue to explain in succession.
The order is 1 → 2 → 3 → 4 →5 ↓
6 → 7 → 8 →9 →10 C.elegans behavior speed
a.Control-N2＞b.N2+Fe3O4＞c.N2+magnetic field＞
d.N2+Fe3O4+magnetic field 48hr＞e.N2+Fe3O4+magnetic field 96hr a.Control-N2
Fig 19. Control-N2 mobility in succession
b.N2+Fe3O4
Fig 20. N2+Fe3O4 mobility in succession
c.N2+magnetic field
Fig 21. N2+magnetic field mobility in succession
d.N2+Fe3O4+magnetic field 48hr
Fig 22. N2+Fe3O4+magnetic field 48hr mobility in succession
e.N2+Fe3O4+magnetic field 96hr
Fig 23. N2+Fe3O4+magnetic field 96hr mobility in succession
3.2 Super-paramagnetic nanoparticles in C.elegans 3.2.1 Super-paramagnetic nanoparticles-Fe3O4 Show in documents before this it is nonpoisonous.
We buy super-paramagnetic nanoparticles from Taiwan Advanced Nanotech Inc Amino-TANBeads / U118 Magnetic beads-Amino terminated (-NH3)
Carboxyl-TANBeads /U128 Product list Magnetic beads-Carboxyl terminated (-COOH)
3.2.2 Optics microscope picture
In order to confirm that really there is FE3O4 that we add in nematode's body We use the optics microscope to observe the state after 3hr feeding.
The following of the result Control
Fig 24. N2 optics microscope picture
The background light source is unavoidable.
C.elegans under the optics microscope is clear and is very easy to observe:
We could find nanoparticle appear in the throat or alimentary canal.
The result is obvious. Place of director's comparatively obvious difference of the prototype.
Experiment
Fig 25. Fe3O4 in C.elegans alimentary canal
3.3 C.elegans SDS-page
Fig 26. C.elegans SDS-page
Function paper of the C.elegans and magnetic field .Exposure of C.elegans to extremely low frequency high magnetic fields induces stress responses. A number of studies suggest that the expression of heat shock protein induced by heat stress is associated with protection. So we follow it.
However, the result is not good. BSA is mark and molecular weight is 69kb. It is lower than 69kb that we can infer the molecule in the red frame bright. 3 groups on the left are all N2+Fe3O4.
On the right is normal N2. We can guess at least that adding Fe3O4 will not influence proteins.
(Whether data behind is it join Fe3O4 can take to gene some influence)
3.4 PCR data
3.4.1 Gene number
For the convenience on the experiment, we number the gene.
Follow-up experiment that it is here that Arabic numeral represents the gene.
1 unc-1 20 unc-52 39 c04e12.7 58 math-33 77 t14f9.3 96 oxi-1
Table.3 Gene number list
3.7.1 .PCR electrophoresis
The following is a result of PCR. It proves the magnetic field will cause the change on the gene.
Base on observation in C.elegans behavior in the front, we know Fe3O4 + the magnetic field synergy a largest influence on C.elegans. Group Control is normal N2. Group Experiment feeds N2 which eats Fe3O4 + magnetic field function. So rough classification is essential .It is not quantitative that PCR behaves. We can fast to find which gene have influence with law this. On the right the
Fig 27.Gene PCR gel 1-25
From 1 to 25, the genes about behavior behave. Seeming to be data has not presented the difference on the result. Of possible .It is a lot of factors will be influenced between primer design and temperature.
Fig 28.Gene PCR gel 26-50
From 26 to 50, later 30 apoptosis was correlated with. Later it was the place where we are relatively interested in. The experiment is brighter than the control and more in most places. This we do not confirmed. The expression of PCR is the showing uncomfortable ration.
Fig 29.Gene PCR gel 51-74
From 50 to 74 .almost are apoptosis related gene and cancer related gene. In contrast, impaired apoptosis may be a significant factor in the etiology of such diseases as cancer, autoimmune disorders, and viral infections.
Fig 30.Gene PCR gel 75-99
From 75 to 99, he left one side of something is that cancer is correlated with. The right one side of something is that oxidation is correlated with. From 1 to99, we find that deduct background he difference of every gene is diminished or has no way to assay. Some are not even believable. So except traditional PCR, we need good tools to prove the difference that the gene expression.
Fig 31.Gene PCR gel 100-109
From 100 to 109, C.elegans and magnetic field function in possible relevant gene studies.
In the research before this, the magnetic field think that correlate with HSPs and C.elegans is studied most frequently usedly in life span .So this group hopes to reappear over forefathers' research.
Fig 32. Repeat gene PCR gel 76-96, L18, L21
We can affirm even more with this picture the magnetic field must influence with the gene relevantly.
L18, L21 on the right is control gene. If the result it is expected, the luminance of group the experiment and group the control are the same. We can confirm 2 genes expression are the same But other groups show different results. Seem to have an effect in magnetic field.
3.5 Real-time PCR data
Real-time PCR is very sensitive. The repetition which wants 3 times is believable. The first data is review, in order to test the stability of data. Because real-time PCR cost is expensive, effective DATA appears as follows. We test all genes first.
3.5.1 Real-time PCR system
The new MyiQ real-time PCR detection system offers an affordable alternative for the detection of common green fluorescent dyes such as FAM and SYBR Green I. This system interfaces directly with the iCycler thermal cycler, offering superior features such as thermal gradient and Peltier-effect driven performance. The MyiQ real-time PCR detection system is a perfect solution for those just getting started with this technology as well as those looking for additional instruments to handle increasing routine assay demands. This system was developed by the same experts at Bio-Rad who have pioneered performance in real-time PCR. The MyiQ delivers the same excellent-quality data as the iCycler iQ real-time detection system.
3.5.1.1 Real –time PCR procedure
Fig 33. Real –time PCR procedure
3.5.2 Gene expression
We use real-time PCR to prove gene expression. The following axes of ordinates of picture (Y axle) Show the disparity that the gene expression. Disparity that the gene expression is a sub number of 2 Gene expression = 2n
Now we give an example in 3.5.3 Fist real-time PCR test(flow) For example1, gene number 31 Y axle is similar -3
Gene expression = 2-3 = 8-1
We can say in this gene number 31 gene expression the experiment is 8 times lower than the control.
So gene number 31 Gene expression was reduced in the mechanism of testing at this moment. And it has reduced by 8 times
For example2, gene number 75 Y axle is similar 14 Gene expression = 214 = 16384
We can say in this gene number 75 gene expresssion in the experiment is 16384 times higher than thecontrol. So gene number 75 Gene expression increased in the mechanism of testing at this moment. And it has increased 16384 times.
3.5.3 Fist time real-time PCR test
control N2
experiment N2+Fe3O4+MF
The axes of ordinates of chart(Y axle) are t △gene expression(the experiment－the control) The axes of horizontal of chart(X axle) are the gene name serial numbers
The result is not believable, because it is only work one time.
The positive number expresses the experiment group ＞ the control.
The negative number expresses the experiment group ＜ the control (data base on Fig 34)
Fig 34. Fist time real-time PCR data test
3.5.4 Real-time PCR data believable The result is believable. It is three time replay.
Real-time PCR is very sensitive, so we must utilize knowledge, experience and effective tool to get rid of useless data. A simple method is to see whether PCR Amp/Cycle and Melt Curve accord with expectancy.
Prove by following 2 Graph. We adopt just with PCR curve intact and sharp Melt Curve. Because data is numerous, we got rid of no influencing and data disorderly. The following data is all covering triply, the credibility is very high
PCR Amp/Cycle Graph for SYBR-490
Fig 35. PCR Amp/Cycle Graph for SYBR-490
Melt Curve Graph for SYBR-490
Fig 36. Melt Curve Graph for SYBR-490
3.5.4.1 Ced family gene expression
Fig 37.Ced real-time PCR data to gene expression
Ceds are very famous for apoptosis research gene . The control is normal N2
The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF)
We can observe that all gene expression increase with magnetic field obviously. Though add Fe3O4 to gene expression has influence, but we can confirm the magnetic field has really influenced gene expression even more.
3.5.4.2 Other apoptosis related gene expression
Fig 38.Other apoptosis related gene real-time PCR data to gene expression
The control is normal N2 The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF) These apoptosis related genes are all very famous.
Take abl-1 as an example:
A very obvious one that adds Fe3O4 to C.elegans is not affected. But gene expression is very obvious under magnetic field function. Magnetic field function disparity is reached 25 = 32 times.
3.5.4. Some cancer related gene expression
Fig 39.Some cancer related gene real-time PCR data to gene expression
The control is normal N2 The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF
We are very sorry about that it is not easy to look over t the chart small very much.
But we can still perceive the magnetic field has really influenced gene expression.
3.6 C.elegans GFP marked
Fig 40. GFP mark signal
From CGC mutant C.elegans, some strain has GFP mark; .liko cbp-1.Cbp-1 has GFP mark in pharyngeal. We culture cbp-1 C.elegans with the magnetic field 96 hr (right) and with no magnetic environment (left).
Fig 41.GFP signal decrease with magnetic field
We find an interesting thing that pharyngeal GFP signal will be reduced acting on continuously with magnetic field. Though CBP-1 can change with the magnetic field, pharyngeal GFP signal is not reporter gene GFP signal. Although the cbp-1 gene expression decrease with magnetic field in our experiment, it can't be proved while making people interesting like this.
3.7 C.elegans mutant behavior ＆ real-time PCR data
Fe3O4 ultra paramagnetic nanoparticles influence gene expression and strengthen the impact on gene expression of the magnetic field. But this ultra paramagnetic nanoparticles has spontaneous magnetic field. In order to distinguish the spontaneous magnetic field and the magnetism field influence. We join 2 kinds of nonpoisonous nanoparticles-Fe2O3, TiO2...Fe2O3andTiO2.diameter are (40nm) slightly biger than Fe3O4 (10nm).
Fe2O3 is compared with Fe4O3; they are all oxides of the iron. The difference is the ultra paramagnetic in Fe3O4 has spontaneous magnetic fields. The same one they are attracted by magnetic field. : They all move with the direction of the magnetic field. Join Fe2O3 in order to prove what is influenced is in the experiment. It was not only a spontaneous magnetic field that was influenced, the more important thing environment magnetic field.
TiO2 is compared with Fe4O3. They are all nonpoisonous nanoparticles. Fe3O4 ultra paramagnetic nanoparticles influence gene expression and strengthen the impact on gene expression of the magnetic field. Nanoparticle influence gene expression or environment magnetic field. This is important thing. TiO2 is useful for riding of the influence of the iron oxide and distinguishing nanoparticles influences.
We request strains from CGC
C.elegans mutant gene CGC strain C.elegans mutant gene CGC strain
unc-30 EW45 unc-119 XA3504
Table 4 . Request strains from CGC
Now we use 3 new mutant strains, ced-3 MT1522, ced-6 MT4970, cbp-1 VC1006 to prove our result.Ced-3 and ced-6 are apoptosis related gene. Cbp-1 is cancer related gene. They are all deficient mutant.Ced-3 and ced-6 is in the same pathway in apoptosis. We find apoptosis deficient mutant is insensitive to magnetic field.
Mutant location genomic environs
Ced-3 MT1522
Ced-6 MT4970
Cbp-1 VC1006
Fig 42 Ced-3, ced-6, cbp-1 mutant location genomic environs
3.7.1 Ced-3 MT1522
3.7.1.1 Ced-3 mutant behavior
Fig 43. Ced-3 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05.Recording mode is as same as before utilizing. We compare normal N2 with ced-3 mutant C.elegans. The result is that ced-3 mutant C.elegans is not sensitive to the magnetic field.
3.7.1.2 Ced-3 mutant real-time PCR data
Fig 44. Ced-3 mutant real-time PCR data to gene expression
The type adds the kind of nanoparticles above the chart (bar1 has no nanoparticle)
It is three time replay. Different display ways before following. : Question that we melt simply.
Whether only the magnetic field will have changes to ced-3 mutant C.elegans. The result is excited, not adding any nanoparticle, (bar1)
Gene expression with magnetic field increase more than 5 times of Gene expression without magnetic field. This result expresses magnetic field and ced-3 positive correlation.Ced-3 is important apoptosis pathway. We can suppose boldly the magnetic field correlate with apoptosis.
The magnetic field causes apoptosis.
The magnetic fields still change gene expression and it is positive correlated. Though seem to add nanoparticles (Fe3O4 Fe2O3 TiO2) reduce the difference. We do not care about this. Because on generally speaking, it contain inside great and complicated problem that are not solved.
3.7.2 Ced-6 MT4970
3.7.2.1 Ced-6 mutant behavior
Fig 45. Ced-6 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05 Recording mode is as same as before utilizing.
we compare normal N2 with ced-6 mutant C.elegans.
Two of their results are similar. The possible reason is as follows 1. Ced-6 mutant damage is not serious
2. Gene relevant degree is not enough 3. Gene pathway not related directly
Behavior datas show ced-6 mutant and magnetic field function have no relation between with themselves. The result accords with the expectancy of apoptosis pathway
No matter how, we can still affirm there is an influence in the magnetic field
3.7.2.2 Ced-6 mutant real-time data
Fig 46. Ced-6 mutant real-time PCR data to gene expression
If C.elegans gene related to phagocytosis mutant in the body, liko ced-6, may escape death.
Except that the passive role with death cell's skeleton of a corpse of phagocyting and is not merely acted and moved around the cell. Even cause the initiative assailant of apoptosis [31].
Ced-3 and ced-6 are not in same pathway , although they are apoptosis related. The result shows the lower gene expression with magnetic field function.
Fig 47.ced-6 apoptosis related pathway
3.7.3 Cbp-1 VC1006
3.7.2.1 Cbp-1 mutant behavior
Fig 48. Cbp-1 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05.
Cbp-1 mutant is compared with N2.
We look cbp-1 mutant C.elegans is more sensitive to the magnetic field instinctively. This kind of method instinctively lacks the scientific idea.
N2 speed under magnetic field function reduces almost 3 mm/min. (N2 vs. N2+MF from 7 to 4) Cbp-1 mutant speed under magnetic field function reduces almost 3 mm/min. (cbp-1 vs. cbp-1+MF from 5.5 to 2.5).
Their disparity is all 3. We can not put the final conclusion that cbp-1 mutant C.elegans is more sensitive to the magnetic field instinctively.
3.7.2.2 Cbp-1 mutant real-time data
Fig 49. Cbp-1 mutant real-time PCR data to gene expression
Over 80% of the cancers known produce, because p53 is lost badly at present.
P53 is important, because it has 3 functions:
1. Order cells not to grow again, is engaged in the work of mending damaged DNA attentively
1. Order cells not to grow again, is engaged in the work of mending damaged DNA attentively