The document summarizes experiments performed from February to August to isolate RNA and protein from small numbers of bacterial cells (106 to 103) and recover bacteria from blood samples. Key experiments included: 1) Isolating protein from E. coli cells using TRI reagent and visualizing via SDS-PAGE, 2) Isolating RNA from E. coli cells using various kits and quantifying, 3) Isolating bacteria from spiked blood samples stored under different conditions, and 4) Detecting isolated RNA from low numbers of E. coli cells using quantitative PCR. The results showed that most bacteria were recovered from the red blood cell fraction, and storage at -20°C led to complete hemolysis and zero bacterial recovery.

1. Sepsis Work Report
February to August
Sonali Uttam
Junio Research Fellow
Dr. Krishnamohan Atmakuri
Principal Investigator
Translational Health Science and Technology Institute
Faridabad, India

2. Objectives
• Isolate RNA and protein from small number of
bacterial cells (106 to 103)
• Recover bacteria from (spiked) blood.
• Isolate bacterial RNA from (spiked) blood.

3. Experiments performed:
1. Protein Isolation from E. coli (106 to 103 cells)
• TRI reagent
• Laemmli sample buffer
2. Visualization of isolated bacterial protein by SDS-PAGE and :
a) Coomassie Staining
b) Silver Staining
3. RNA isolation form E. coli (106 to 103 cells)
a) TRIzol/ TRI reagent
b) RNASnap
c) Nucleosping RNA XS Kit
d) Nucleospin TRIPrep kit
e) PicoPure Kit
4. RNA isolation of bacteria present in blood
• Qiagen UCP Blood Pathogen Kit

4. Experiments performed (contd.)
5. Quantification of isolated RNA
a) NanoDrop
b) Qubit High Sensitivity RNA Assay
6. Isolation of bacteria from blood (spiked and stored at various storage
conditions)
7. Detection and estimation of RNA isolated from 103 and 102 E. coli cells,
using two step qPCR (SYBR)
8. Isolation of whole RNA from blood spiked with E. coli cells.

5. Bacterial cell pellet (washed once with PBS)
Re-suspend pellet in 1ml TRI reagent by repeated pipetting and vortexing.
Incubate, RT, 5-10 min
Add 250ul chloroform & shake vigorously. Allow to stand for 2-3 min, RT.
(Phase separation)
RNA/Protein Isolation using TRI reagent
12000 x g; 4°c; 15 min
Organic Phase: Proteins
Inter-phase: DNA
Aqueous phase: RNA

6. Organic phase and inter-phase
Add 300ul 100% EtOH, mix well
Collect Supernatant in fresh tube
Add 1.5ml Isopropanol, incubate for 10-15 min, RT
12000 x g,4°c,15 min
Wash pellet 3 times with 0.3M GnHCl/95% EtOH
Wash once with 2ml absolute EtOH
Dissolve pellet in 1X Laemmli’s buffer, store at -20°C
Pellet: DNA
Protein isolation

7. Exp-2a: Proteins isolated from 106 and 103 E. coli cells
SDS-PAGE gel, Coomassie Stain
Sample Buffer TRI Reagent

8. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Exp-2b: Proteins isolated from 106 bacterial cells using TRIzol/TRI reagent
Silver stain of SDS-PAGE gel
SB: Sample Buffer TRI: TRI reagent

9. Sample Buffer TRI Reagent
Proteins isolated from 106 and 103 bacterial cells using TRI reagent

10. 3 March 16
SB: Sample Buffer TRI: TRI reagent
Proteins isolated from 106 and 103 bacterial cells using TRI reagent

11. Protein isolation by NucleoSpin TRIprep kit and TRI reagent
11 May 2016

12. Aqueous phase collected in a fresh MCT
Add 10µg of Glycogen
Add 500µl of Isopropanol, Invert mix very gently
Incubate at -20°C for over night
12000 x g,4°c, 15 to 20 min
Add 1ml of 75% EtOH, vortex briefly
Dry the pellet in laminar air flow hood for 5min
Dissolve pellet in Nuclease free water(Sigma), Keep tubes at 55°C for 10min.
Immediately, cool on ice. Pipette up and down 3-4 times.
Pellet: DNA
Discard supernatant. Dry the pellet in laminar air flow hood for 5min
7600 x g,4°c, 10 min
RNA isolation

13. RNA isolated from 106 bacterial cells using TRIzol/TRI reagent

14. A B C D
RNA isolated from 106 and 103 bacterial cells using TRIzol/TRI reagent

15. 1.5ml culture (1) 750.0 493.1 1.9 1.14
1.5ml culture (2) 690.0 438.4 1.98 0.68
106 cells (1) 18.2 24.7 1.7 0.19
106 cells (2) 17.7 17.6 1.84 0.15
103 cells (1)
Low-Out of
range 10.7 1.51 0.18
103 cells (2) 0.4 17.5 1.61 0.2
Qubit NanoDrop
Sample
Concentration
(ng/µl)
Concentration
(ng/µl)
A260/280 A260/230
Quantification of RNA isolated by TRI Reagent

16. 3000 bp
1000 bp
500 bp
Exp-3b: RNA isolation by RNAsnap Method
(Pellet lysed in 100µl RNA extraction buffer (95% Formamide, 18mM EDTA and
0.025% SDS)
Gel: 1.2 % Agarose in 1X TAE buffer
EtBr: 20µl of 1mg/ml stock in 50ml of gel
Voltage: 5V/cm
Sample:
10µl of RNA sample in 2X Formaldehyde dye (2XF).
Total volume loaded : 20µl
Before loading samples were incubated at 95ºC
with 2X formaldehyde loading dye, for 5min and
snap frozen on ice.
Ref. for protocol: Stead et al., RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA from
bacteria, Oxford journals (2012)

17. Qubit NanoDrop
Sample
Concentration
(ng/µl)
Concentration
(ng/µl)
A260
/280
A260/
230
1ml Culture 1133.3 1037.7 1.69 5.95
700µl culture 818.7 744.3 1.68 4.23
105cells (1) 0.5 6.3 1.71 3.73
105cells (2) 0.5 -7.8 -6.32 -37.82
Quantification of RNA isolated by RNASnap Method

18. Source: http://www.mn-
net.com/Products/DNAandRNApurification/RNA/NucleoSpinTriPrep/tabid/11113/language/en-US/Default.aspx
RNA , DNA and Protein Isolation by NucleoSpin TRIprep Kit

19. Dye front
1% Agarose gel, TAE, 5V/cm, 20µl was loaded in 2XF(4µl) dye after incubation at 55°C, 5min
Short run (~10min)
3kbp
RNA isolation by NucleoSpin TRIprep

20. RNA isolation by
NucleoSpin RNA XS Kit:
WorkFlow
The funnel shaped thrust ring
of NucleoSpin® RNA XS
column is designed to hold a
silica membrane of very small
diameter, in order to enhance
the RNA extraction from 10 to
1000 cells
Source: http://www.mn-net.com/Products/DNAandRNApurification/RNA/NucleoSpinRNAXS/tabid/10643/language/en-
US/Default.aspx
Source: http://www.mn-
net.com/Products/DNAandRNApurification/RNA/NucleoSpinRNAXS/tabid/10643/languag
e/en-US/Default.aspx

21. Dye front
1% Agarose gel, TAE runnig buffer, 5V/cm, 20µl was loaded in 2XF(4µl) dye after incubation at
55°C, 5min
Short run (~10min)
3kbp
RNA isolation by NucleoSpin XS

22. RNA isolation by PicoPure Kit : Workflow
Source: https://tools.thermofisher.com/content/sfs/manuals/1268200.pdf

23. Nucleic extraction using Blood Pathogen Kit (Qiagen) : work flow
Source: https://tools.thermofisher.com/content/sfs/manuals/1268200.pdf
Whole blood
Plasma

24. Add Qubit working solution for a total volume of 200µl
Exp-5b: RNA Estimation by Qubit
(1 x n) µl
(199 x n) µl Working
solution
Buffer
Reagent
Figures adapted from Qubit® RNA HS Assay Kits, User guide, ThermoFisher Scientific

25. Exp 6: Isolation of bacteria from spiked blood
• Phase separation
• Centrifugation
• HiSep and centrifugation
• Removal of RBCs
• RBC Lysis buffer
• 5µm filter
• Storage condition of spiked blood
• Room temperature, 0days
• 4°C for 0, 4, 7 and 10 days
• 4°C for 0 and 5 days
• -20°C for 5 days

26. Check OD (1ml)
Serial
Dilutions
(100+900)µl
10-3 , 10-4 and 10-5
100µl
Make Serial Dilutions in cold PBS,
According to OD, calculate
required volume of dilution
containing 104 cells
Add calculated volume spike to 1.5ml blood
1.5ml spiked
blood aliquot
100 and 10-1
50µl 100µl
X2 X2 X2
X2
0.65 OD
O/N culture (inoculated
from glycerol stock)
Kept on Ice for
105 min
•Pellet down 2ml culture,
(8000xg 5min, 4°C)
•Wash with PBS,
•Resuspend in 2ml PBS
12:30pm
Spiking blood

27. Spiked blood aliquots
Tube C (1.5ml)
Tube B (1.5ml)
Tube A (1.5ml)
Plated 50µl (100)
and 100µl (10-1)
DAY 0
Stored at 4°C, 5days
Stored at -20°C, 5 days
Storing spiked aliquot of blood

28. 1000 x g,
4°C,
5min
Phase
Separation
Phase Separation
Plasma
RBC fraction
1. Using centrifugation
2. Using HiSep and centrifugation
HiSep
Blood
400 x g,
20°C,
30min
Accel: 3,
Brake: 1
Plasma
Inter-phase
HiSep
RBC fraction

29. RBC fraction Add 1ml RBC Lysis Buffer
Supernatant
12000 xg ,5min, 4°C
Pellet
(re-suspend in 200µl PBS)
100µl
Removal of RBC using RBC lysis buffer
Incubate at RT / 4°C for 30min

30. 1.5ml spiked blood)
1ml
Rotate,
10 min,
4°C
PBS layer
RBC layer
200xg,
2min, 4°C
200xg,
2min, 4°C
Added 2ml cold PBS
Washing RBC pellet with PBS, followed by filtration

31. PBS layer
RBC layer
Add 2ml cold PBS
Filter through 5µm filter disk
Without positive pressure
Residue on filter disk Filtrate
Washed Filter disk in 5ml PBS
2.10ml
100µl
50µl
X2
X2
50µl X2
50µl/plate
Pre-wet
filter disk
with 1ml
PBS

32. Results:
•Majority of the bacteria accumulated in the RBC fraction.
•Phase separation using HiSep led to the accumulation of bacteria in inter-phase,
HiSep layer as well as the RBC fraction.
•The recovery of bacteria significantly dropped on storage at 4°C.
•Blood sample stored at -20°C for five days was completely hemolysed and the
recovery of bacteria was 0%
• 5micron filter disk was unsuccessful is retaining RBCs with and without positive
pressure applied.

33. Whole blood 10 times diluted blood blood
10 times diluted blood , Magnified
RBCs
Red arrows( )indicate
Shrunk RBCs (on Crenation)
Observation of Blood fractions at various stages of filtration, under a light microscope

34. Filtrate
PBS wash: used to wash the
membrane after filtration
Such large cells were observed
These lines of droplets/cells are seen
because the cover-slip was dragged
after being placed on the slide.
Abundant RBCs are seen in the filtrate,
indicating that 5micron filter was not
effective in separating RBCs from the
diluted blood

35. -20°C, 5days
Storage at -20°C:
Complete hemolysis of blood was observed.
Zero Percent recovery.
A B
A: Stored at 4°C, 5 days
B: Stored at -20°C, 5days
No RBC pellet
obtained in tube B,
after dilution of
blood in PBS and
centrifugation

36. 0
20
40
60
80
100
120
Spiked Day 0
%viability
Recovery on Day 0
0
20
40
60
80
100
120
Day 0 Day 4 Day 7
%viability
Storage period
Recovery after storage on 4°C
without HiSep
With HiSep
Percentage recovery vs storage at 4°C

37. Workflow of qPCR experiment
RNA isolation by TRI reagent, dissolve pellet in 10µl NFW(Sigma)
Quantification of RNA by Nanodrop (optional)
cDNA synthesis ( Verso kit)
Real-time PCR using SYBR chemistry (DYNAMO color flash)
Analysis of Data

38. cDNA synthesis
Component Volume (µl) for 1
reaction
Volume (µl) for n+0.5
reactions
5X Buffer 4 4(n+0.5)
dNTPmix 2 2(n+0.5)
Random hexamer (RH) 1 1(n+0.5)
RT enhancer 1 1(n+0.5)
Verso Enzyme 1 1(n+0.5)
RNA x variable
Water* (DEPC treated) 20 - (9+x) {20 - (9+x)}(n+0.5)
Total 20 20(n+0.5)
Step Temperature(°C) Time (min) No. of cycles
cDNA synthesis 42 30 1
Inactivation 95 2 1
Hold 4 infinity 1
Reaction set up:

39. Real time PCR
Component Volume (µl) for 1 reaction Volume (µl) for n+1 reaction
Dynamo 2X Buffer 10 10(n+1)
Forward primer (10µM stock)(FP) 0.4 0.4(n+1)
Reverse primer (10µM stock)(RP) 0.4 0.4(n+1)
cDNA x variable
Water* DEPC treated 20 - (10.8+x) {20 - (10.8+x)}(n+1)
Total 20 20(n+1)
Step Temperature
(°C)
Time No. of
cycles
Amplification 95 7 min 1
95 10 sec
4060 30
Melt Curve 95 1min 1
55 30 sec 1
95 30 sec 1
Reaction set up:
Thermal Profile:

40. Preparation of standard curve of cDNA concentrations vs CT value
Concentration
(pg of cDNA)
Technical
replicate
1
Technical
replicate
2
Mean CT
value
Log of amount of
cDNA (pg)
25,000 23.68 23.63 23.66 4.40
15,000 24.76 24.83 24.80 4.18
5,000 26.49 26.7 26.60 3.70
1,000 29.48 29.25 29.37 3.00
150 31.72 31.87 31.80 2.18
50 33.35 33.64 33.50 1.70
15 35.33 35.55 35.44 1.18
5 36.95 37.08 37.02 0.70
1.5 37.36 37.95 37.66 0.18
0 (NTC) 37.87 34.98 36.43 ND
NTC: No template control; ND: Not defined

41. y = -3.3984x + 39.095
R² = 0.9935
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
0.00 1.00 2.00 3.00 4.00 5.00
CT
Log of amount of cDNA (pg)
Standard curve Ct value vs. amount of cDNA used in a qPCR reaction
Mean CT value
Linear (Mean CT
value)
Efficiency of qPCR = 96.92%
Efficiency = -1+10(-1/slope)

42. Using qPCR to detect RNA isolated (by TRI reagent)
from less than 200 E.coli cells
NTC: No template control
Sample CT value Mean CT
NanoDrop
(ng/µl)
A260/280 A260/230
Amount of
cDNA
using
standard
curve
NTC 39.78
39.78
NTC No Ct
25 ng
RNA
T1 21.07
20.93 1110.5 1.94 1.62
T2 20.79
25 ng
RNA
T1 22.77
22.83 1110.5 1.94 1.62
T2 22.89
25 cells
B1 30.36
30.04
11.6 1.6 0.46
1 ng
B2 29.72 10.4 1.73 0.33
146
cells
B1 28.44
27.00
27.7 1.49 0.49
2.3 ng
B2 25.55 21.8 1.56 0.41

43. Future Experiments:
•Isolate RNA from spiked blood samples.
•Obtain enriched prokaryotic RNA from total RNA isolated
from spiked blood. This is to be done by MICROEnrich kit.
•Isolate RNA from nenotal / cord blood. Determine the yield
according to the amount of TRI reagent used, storage time
and temperature.